Mutant Herpes Simplex Viruses Comprising Nucleic Acid Encoding A Nitroreductase

ABSTRACT

An herpes simplex virus wherein the herpes simplex virus genome comprises nucleic acid encoding a nitroreductase (NTR) is disclosed. Disclosed herpes simplex viruses are indicated to be useful in the treatment of cancer which may involve gene directed enzyme prodrug therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/579,606, filedMay 16, 2006, entitled “Mutant Herpes Simplex Viruses Comprising NucleicAcid Encoding A Nitroreductase,” which is a '371 national stage patentapplication of PCT/GB2004/004851 filed Nov. 17, 2004, entitled“Oncolytic Herpes Simplex Virus Encoding A Heterologous Nitroreductase,”which claims priority to United Kingdom Patent Application No. GB0326798.6 filed Nov. 13, 2004, entitled “Methods for Generating MutantVirus,” each of which is incorporated herein in its entirety byreference.

FIELD OF THE INVENTION

The present invention relates to mutant herpes simplex viruses whereinthe herpes simplex virus genome comprises nucleic acid encoding anitroreductase.

BACKGROUND TO THE INVENTION Herpes Simplex Virus

The herpes simplex virus (HSV) genome comprises two covalently linkedsegments, designated long (L) and short (S). Each segment contains aunique sequence flanked by a pair of inverted terminal repeat sequences.The long repeat (RL or R_(L)) and the short repeat (RS or R_(S)) aredistinct.

The HSV ICP34.5 (also γ34.5) gene, which has been extensivelystudied^(1,6,7,8), has been sequenced in HSV-1 strains F⁹ and syn17+³and in HSV-2 strain HG52⁴ One copy of the ICP34.5 gene is located withineach of the RL repeat regions. Mutants inactivating both copies of theICP34.5 gene (i.e. null mutants), e.g. HSV-1 strain 17 mutant 17162(HSV1716) or the mutants R3616 or R4009 in strain F⁵, are known to lackneurovirulence, i.e. be a virulent, and have utility as both genedelivery vectors or in the treatment of tumours by oncolysis. HSV-1strain 17 mutant 1716 has a 759 bp deletion in each copy of the ICP34.5gene located within the BamHI s restriction fragment of each RL repeat.

ICP34.5 null mutants such as HSV1716 are, in effect, first-generationoncolytic viruses. Most tumours exhibit individual characteristics andthe ability of a broad spectrum first generation oncolytic virus toreplicate in or provide an effective treatment for all tumour types isnot guaranteed.

HSV1716 is described in EP 0571410 and WO 92/13943 and has beendeposited on 28 Jan. 1992 at the European Collection of Animal CellCultures, Vaccine Research and Production Laboratories, Public HealthLaboratory Services, Porton Down, Salisbury, Wiltshire, SP4 0JG, UnitedKingdom under accession number V92012803 in accordance with theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure(herein referred to as the ‘Budapest Treaty’).

Nitroreductase Prodrug Activation

Enzyme prodrug therapy is based on the enzymatic activation of a nontoxic or low toxicity prodrug to a form that is considerably morecytotoxic. The activation may involve enzymatic reduction of the prodrugto a cytotoxic reduced form.

The E. coli nitroreductase enzyme (NTR) has been proposed for use ingene-directed enzyme prodrug therapy (GDEPT) as an activating enzyme fornitroaromatic prodrugs of the dinitrobenzamide class¹⁶ . E. coli NTR isa homodimeric enzyme with two active sites and is the oxygen insensitiveenzyme from E. coli (the nfsB gene product). It has the ability toreduce a wide range of nitro-containing compounds such as nitrofurazone(to the hydroxylamines) and quinones such as menadione (to the quinols).It is specifically inhibited by the irreversible inhibitor dicoumarol.

The ability of NTR to reduce aromatic nitro groups to the correspondinghydroxylamine (and possibly amine) derivatives has been proposed forcancer chemotherapy mainly with the dinitrobenzamide class of prodrugs.The 5-aziridin-1-yl-2,4-dinitrobenzamide CB1954 (CAS Registry number21919-05-1) is one such prodrug which has been studied as a prodrug forGDEPT with NTR¹⁶.

Cyclic and acyclic nitroaryl phosphoroamide mustard analogues have alsobeen shown to be activated by E. coli NTR¹⁷. The acyclic 4-nitrobenzylphosphoramide mustard showed 167,500× selective cytotoxicity towardnitroreductase-expressing V79 cells with an IC₅₀ as low as 0.4 nM whichis about 100× more active and 27× more selective than CB1954.

Recombinant adenovirus and recombinant retrovirus¹⁰ expressingnitroreductase have been constructed for use with the prodrug CB1954with the intention of providing a treatment for cancer. The recombinantvirus is not oncolytic and relies on gene directed enzyme-prodrugtherapy to achieve tumour cell kill.

SUMMARY OF THE INVENTION

The inventors have determined that herpes simplex virus having aninactivating mutation in the RL1 locus, more specifically a mutationwhich inactivates the function of the ICP34.5 gene product, such thatthe herpes simplex virus does not produce a functional ICP34.5 geneproduct and is non-neurovirulent, can be used in the delivery to a cellof a gene encoding a gene product useful in targeted tumour therapy.

The inventors have provided a novel second generation oncolytic mutantHSV. The genome of this mutant HSV comprises the heterologous (i.e.non-HSV originating) E. coli nitroreductase protein coding sequenceinserted at one or each ICP34.5 locus, disrupting the ICP34.5 proteincoding sequence such that the ICP34.5 gene is non-functional and cannotexpress a functional ICP34.5 gene product. The generated HSV is capableof expressing the E. coli nitroreductase gene product under control ofthe inserted promoter. This virus thus has the oncolytic activity of HSVstrain 17 mutant 1716 and can be used in gene directed enzyme-prodrugtherapy (GDEPT) and has shown significantly enhanced tumour cell killingin vitro and in vivo when used with the prodrug CB1954. The mutant virusis designated HSV1716/CMV-NTR/GFP (also called HSV1790).

HSV1716/CMV-NTR/GFP is an engineered herpes simplex virus ICP34.5 nullmutant which expresses the nitroreductase (NTR) gene. This virusprovides for enhanced virus induced tumour cytotoxicity. It combines NTRtransgene delivery and CB 1954 prodrug treatment with theproliferation-specific, lytic capacity of HSV1716.

The heterologous nitroreductase polypeptide expressed by an herpessimplex virus according to the present invention may be useful in genedirected enzyme-prodrug targeting techniques for tissue specificdelivery of active pharmaceutical agents derived by nitroreductasedependent activation of the NTR prodrug.

In vivo, the inventors have demonstrated that the nitroreductase gene,when introduced by HSV1716/CMV-NTR/GFP into mouse gliomal xenograftmodels in combination with the prodrug CB1954, results in delay intumour growth and in oncolysis. Administering both theHSV1716/CMV-NTR/GFP virus and CB1954 prodrug in combination was observedto produce a greater effect than either virus or prodrug alone, i.e. thecombination exhibits a synergistic effect.

The results demonstrate that the combination of oncolytic HSV therapywith gene therapy directed nitroreductase/prodrug treatment provides aneffective means of tumour cell kill and tumour growth reduction andthereby a treatment for tumour.

Combining herpes simplex virus HSV1716-mediated oncolysis withnitroreductase gene transfer has yielded results exhibiting a surprisingsynergy and provides a novel therapeutic strategy for treatment oftumours of all kinds.

HSV1716/CMV-NTR/GFP has been deposited in the name of CrusadeLaboratories Limited having an address at Department of NeurologySouthern General Hospital 1345 Govan Road Govan Glasgow G51 5TF Scotlandon 5 Nov. 2003 at the European Collection of Cell Cultures (ECACC),Health Protection Agency, Porton Down, Salisbury, Wiltshire, SP4 0JG,United Kingdom under accession number 03110501 in accordance with theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure(herein referred to as the ‘Budapest Treaty’).

Accordingly, the present invention relates to a herpes simplex virus,wherein the herpes simplex virus genome comprises a nucleic acidsequence encoding a nitroreductase. The herpes simplex virus may also benon-neurovirulent.

At its most general the present invention relates to an herpes simplexvirus wherein the herpes simplex virus genome comprises nucleic acidencoding a nitroreductase.

According to one aspect of the present invention there is provided anherpes simplex virus wherein the herpes simplex virus genome comprisesnucleic acid encoding an heterologous nitroreductase (NTR).

Said nucleic acid may encode an E. coli NTR and may comprise, consist ofor include the nucleic acid sequence of SEQ ID No. 2. Alternatively thenucleic acid may have at least 60% sequence identity to SEQ ID No. 2.Said degree of sequence identity may alternatively be one of at least70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% provided the polypeptide orprotein encoded by such nucleic acid has a nitroreductase function.Identity of sequences is determined across the entire length of a givennucleotide sequence. Where sequences are of different length, sequenceidentity of the shorter sequence is determined over the entire length ofthe longer sequence.

Said nucleic acid encoding NTR may be selected by its ability tohybridise to the nucleic acid of SEQ ID No. 2, or its complement, underhigh stringency conditions.

The genome of said herpes simplex virus may further comprise aregulatory sequence operably linked to said nucleic acid encoding NTR,wherein said regulatory sequence has a role in controlling transcriptionof said nucleic acid.

The nucleic acid encoding NTR may be located in at least one RL1 locusof the herpes simplex virus genome. Suitably it may be located in, oroverlap, at least one of the ICP34.5 protein coding sequences of theherpes simplex virus genome. The nucleic acid may be located in both(this will usually be all) copies of the RL1 locus or ICP34.5 proteincoding sequence.

The herpes simplex virus is preferably a mutant and may be a mutant ofHSV-1 or HSV-2, more preferably of one of HSV-1 strains 17, F or HSV-2strain HG52. The herpes simplex virus may be a further mutant of HSV-1strain 17 mutant 1716.

In certain arrangements the herpes simplex virus may be a gene specificnull mutant, such as an ICP34.5 null mutant.

In other arrangements the herpes simplex virus may lack at least oneexpressible ICP34.5 gene.

In yet another arrangement the herpes simplex virus may lack only oneexpressible ICP34.5 gene.

In yet another arrangement the herpes simplex virus may benon-neurovirulent.

In herpes simplex viruses of the present invention the nucleic acidencoding the NTR may form part of a nucleic acid cassette permanentlyintegrated in the herpes simplex virus genome, said cassette comprisingnucleic acid encoding:

(a) said nucleic acid encoding NTR; and nucleic acid encoding:

(a) a ribosome binding site; and

(b) a marker,

wherein the nucleic acid encoding NTR is arranged upstream (5′) of theribosome binding site and the ribosome binding site is arranged upstream(5′) of the marker, wherein said ribosome binding site has a role incontrolling transcription of said marker.

A regulatory nucleotide sequence may be located upstream (5′) of thenucleic acid encoding NTR, wherein the regulatory nucleotide sequencehas a role in controlling and regulating transcription of the nucleicacid encoding the NTR^(X) and hence expression of the resultingtranscript and polypeptide. The regulatory sequence may compriseselected promoter or enhancer elements known to the person skilled inthe art, e.g. the CytoMegalovirus (CMV) promoter. Suitably theregulatory sequence may be constitutive or inducible.

The components of the cassette are preferably arranged in apredetermined order.

In one preferred arrangement, the nucleic acid encoding NTR is arrangedupstream (i.e. 5′) of the ribosome binding site and the ribosome bindingsite is arranged upstream (i.e. 5′) of the marker. During transcriptiona single transcript may be produced from the cassette comprising a firstcistron comprising nucleic acid encoding NTR (e.g. an mRNA transcript)and a second cistron comprising nucleic acid encoding the marker whereinthe ribosome binding site is located between the cistrons.

A transcription product of this cassette may be a bi- or poly-cistronictranscript comprising a first cistron encoded by the nucleic acidencoding NTR and a second cistron encoding the marker nucleic acidwherein the ribosome binding site is located between said first andsecond cistrons.

In another arrangement, the nucleic acid encoding the NTR may bearranged upstream (i.e. 5′) of a first regulatory nucleotide sequenceand the first regulatory nucleotide sequence is arranged upstream (i.e.5′) of the marker.

The cassette may disrupt a protein coding sequence of the herpes simplexvirus genome resulting in inactivation of the respective gene product.

One suitable ribosome binding site comprises a ribosome entry sitepermitting entry of a ribosome to the transcribed mRNA encoded by thenucleic acid of the cassette such that the ribosome binds to thetranslation start signal. Preferably, the ribosome entry site is aninternal ribosome entry site (IRES), more preferably anencephalomyocarditis virus IRES, permitting cap-independent initiationof translation. The IRES thus enables translation of a coding sequencelocated internally of a bi- or poly-cistronic mRNA, i.e. of a cistronlocated downstream of an adjacent cistron on a single transcript.

Preferably the marker is a defined nucleotide sequence coding for apolypeptide which can be expressed in a cell line (e.g. BHK cells)infected with mutant herpes simplex virus into which the cassette hasbeen recombined. The function of the marker is to enable identificationof virus plaques containing mutant virus transformed with the cassette.

The marker is preferably a detectable marker, more preferably anexpressible marker polypeptide or protein comprising at least the codingsequence for the selected polypeptide or protein. The nucleic acidencoding the marker may further comprise regulatory sequence upstreamand/or downstream of the coding sequence having a role in control oftranscription of the marker mRNA. Preferred markers include the GreenFluorescent Protein (GFP) protein coding sequence or gene, preferablythe enhanced Green Fluorescent Protein (EGFP) protein coding sequence orgene.

In other arrangements the marker may comprise a defined nucleotidesequence which can be detected by hybridisation under high stringencyconditions with a corresponding labelled nucleic acid probe, e.g. usinga fluorescent- or radio-label.

The cassette may also comprise nucleic acid encoding a polyadenylation(“polyA”) sequence, which sequence is preferably located downstream (3′)of the nucleic acid encoding the marker. One preferred polyA sequence isthe Simian Virus 40 (SV40) polyadenylation sequence. The preferredlocation of the polyA sequence within the cassette is immediatelydownstream (i.e. 3′) of the marker.

Mutant herpes simplex viruses of the present invention may be generatedby site directed insertion of a nucleic acid cassette into the viralgenome, more preferably by homologous recombination. However, theviruses of the invention are not limited to Herpes simplex virusesobtained in this way.

In other aspects of the present invention herpes simplex virusesaccording to the present invention are provided for use in a method ofmedical treatment. Suitably they are provided for use in the treatmentof disease. Preferably they are provided for use in the treatment ofcancer. Suitably they may be provided for use in the oncolytic treatmentof cancer/a tumour. The use of herpes simplex viruses according to thepresent invention in the manufacture of a medicament for the treatmentof cancer is also provided.

In another aspect of the present invention medicaments comprising herpessimplex virus mutants according to the present invention for use inoncotherapy and methods of treating tumours comprising administering toa patient in need of treatment an effective amount of a mutant HSV or amedicament comprising or derived from such HSV are also provided.Methods of lysing or killing tumour cells in vitro or in vivo comprisingthe step of administering to a patient in need of treatment an amount ofan Herpes simplex virus according to the present invention are alsoprovided.

A medicament, pharmaceutical composition or vaccine comprising an Herpessimplex virus according to the present invention is also provided. Themedicament, pharmaceutical composition or vaccine may further comprise apharmaceutically acceptable carrier, adjuvant or diluent. Pharmaceuticalcompositions or vaccines may further comprise an NTR prodrug.

The present invention may also include the following aspects which maybe provided in combination with any of the other aspects and featuresdescribed.

According to another aspect of the present invention a herpes simplexvirus is provided, wherein the genome of said virus comprises a nucleicacid sequence encoding an heterologous nitroreductase (NTR) in at leastone of the long repeat regions (R_(L)).

According to another aspect of the present invention a herpes simplexvirus is provided, wherein the genome of said virus comprises a nucleicacid sequence encoding an heterologous nitroreductase (NTR) and whereinthe herpes simplex virus is non-neurovirulent.

A composition comprising a herpes simplex virus of the invention may beprovided in combination with an NTR prodrug. The NTR prodrug may beCB1954.

According to another aspect of the present invention a herpes simplexvirus for use in the treatment of a tumour is provided, wherein thegenome of said virus comprises a nucleic acid sequence encoding anheterologous nitroreductase in at least one of the long repeat regions(R_(L)).

According to another aspect of the present invention a herpes simplexvirus for use in the treatment of a tumour is provided, wherein thegenome of said virus comprises a nucleic acid sequence encoding anheterologous nitroreductase and wherein the herpes simplex virus isnon-neurovirulent.

According to another aspect of the present invention a herpes simplexvirus is provided, wherein the genome of said virus comprises a nucleicacid sequence encoding an heterologous nitroreductase in at least one ofthe long repeat regions (R_(L)), for use, in combination with an NTRprodrug, in the treatment of a tumour.

According to another aspect of the present invention a herpes simplexvirus is provided, wherein the genome of said virus comprises a nucleicacid sequence encoding an heterologous nitroreductase and wherein theherpes simplex virus is non-neurovirulent, for use, in combination withan NTR prodrug, in the treatment of a tumour.

According to another aspect of the present invention a kit of parts isprovided comprising a first container having a quantity of an herpessimplex virus of the invention and a second container having a quantityof an NTR prodrug.

In another aspect the use of a herpes simplex virus, wherein the genomeof said virus comprises a nucleic acid sequence encoding an heterologousnitroreductase in at least one of the long repeat regions (R_(L)), inthe manufacture of a medicament for the treatment of a tumour is alsoprovided.

In another aspect the use of a herpes simplex virus, wherein the genomeof said virus comprises a nucleic acid sequence encoding an heterologousnitroreductase and wherein the herpes simplex virus isnon-neurovirulent, in the manufacture of a medicament for the treatmentof a tumour is also provided.

In another aspect the use in the manufacture of a medicament for thetreatment of a tumour of a herpes simplex virus, wherein the genome ofsaid virus comprises a nucleic acid sequence encoding an heterologousnitroreductase in at least one of the long repeat regions (R_(L)), andan NTR prodrug is also provided.

In another aspect the use in the manufacture of a medicament for thetreatment of a tumour of a herpes simplex virus, wherein the genome ofsaid virus comprises a nucleic acid sequence encoding an heterologousnitroreductase and wherein the herpes simplex virus isnon-neurovirulent, and an NTR prodrug is also provided.

In another aspect the use of a herpes simplex virus, wherein the genomeof said virus comprises a nucleic acid sequence encoding an heterologousnitroreductase in at least one of the long repeat regions (R_(L)) in themanufacture of a first medicament for administering sequentially orsimultaneously with a second medicament comprising an NTR prodrug in thetreatment of a tumour is also provided

In another aspect the use of an NTR prodrug in the manufacture of afirst medicament for administering sequentially or simultaneously with asecond medicament comprising a herpes simplex virus, wherein the genomeof said virus comprises a nucleic acid sequence encoding an heterologousnitroreductase in at least one of the long repeat regions (R_(L)), inthe treatment of a tumour is also provided.

In another aspect the use of an NTR prodrug in the manufacture of afirst medicament for administering sequentially or simultaneously with asecond medicament comprising a herpes simplex virus, wherein the genomeof said virus comprises a nucleic acid sequence encoding an heterologousnitroreductase and wherein the herpes simplex virus isnon-neurovirulent, in the treatment of a tumour is also provided.

In another aspect the use of a herpes simplex virus, wherein the genomeof said virus comprises a nucleic acid sequence encoding an heterologousnitroreductase and wherein the herpes simplex virus isnon-neurovirulent, in the manufacture of a first medicament foradministering sequentially or simultaneously with a second medicamentcomprising an NTR prodrug, in the treatment of a tumour is alsoprovided.

The time period between sequential administrations may be such that theherpes simplex virus and NTR prodrug may interact in the body to producean active pharmaceutical agent in situ. Preferred time periods may beless than 15 minutes, less than one hour, two hours, three hours, fourhours, five hours or six hours, twelve hours, twenty four hours, fortyeight hours, one week or two weeks. Either the herpes simplex virus orNTR prodrug may be administered first.

In another aspect a method of treatment of a tumour is providedcomprising the steps of:

-   -   (i) administering to a patient in need of treatment a herpes        simplex virus, wherein the genome of said virus comprises a        nucleic acid sequence encoding a nitroreductase in at least one        of the long repeat regions (R_(L)); and    -   (ii) administering to said patient a therapeutically effective        amount of an NTR prodrug.

In another aspect a method of treatment of a tumour is providedcomprising the steps of:

-   -   (i) administering to a patient in need of treatment a herpes        simplex virus, wherein the genome of said virus comprises a        nucleic acid sequence encoding a nitroreductase and wherein the        herpes simplex virus is non-neurovirulent; and    -   (ii) administering to said patient a therapeutically effective        amount of an NTR prodrug.

In the methods of treatment said herpes simplex virus is preferablycapable of killing tumour cells, e.g. by oncolysis.

In aspects of the invention involving an NTR prodrug, one preferredprodrug is CB1954.

In another aspect a method of expressing in vitro or in vivo anitroreductase is provided, said method comprising the step of infectingat least one cell or tissue of interest with a herpes simplex virus,wherein the genome of said virus comprises a nucleic acid sequenceencoding a heterologous nitroreductase in at least one of the longrepeat regions (R_(L)), said nitroreductase operably linked to atranscription regulatory sequence.

In another aspect a method of expressing in vitro or in vivo anitroreductase is provided, said method comprising the step of infectingat least one cell or tissue of interest with a non-neurovirulent herpessimplex virus, wherein the genome of said virus comprises a nucleic acidsequence encoding a heterologous nitroreductase, said nitroreductaseoperably linked to a transcription regulatory sequence.

Herpes simplex viruses of the invention having nucleic acid encoding anheterologous nitroreductase in at least one of the long repeat regions(R_(L)) of the HSV genome preferably have said nucleic acid in each ofthe long repeat regions of the HSV genome. Two long repeat regions areusually present in the HSV genome.

The NTR nucleotide sequence may encode a full length transcript orpolypeptide (i.e. comprise the complete NTR protein coding sequence).Alternatively, provided the polypeptide product retains nitroreductaseactivity, the NTR nucleotide sequence may comprise one or more fragmentsof the full length sequence respectively coding for a fragment of thefull length transcript or a truncated polypeptide.

A fragment may comprise a nucleotide sequence encoding at least 10% ofthe corresponding full length sequence, more preferably the fragmentcomprises at least 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or99% of the corresponding full length sequence. Preferably, the fragmentcomprises at least, i.e. has a minimum length of, 20 nucleotides, morepreferably at least 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 19002000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100,3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900 or 4000 nucleotides. Thefragment may have a maximum length, i.e. be no longer than, 20nucleotides, more preferably no longer than 30, 40, 50, 100, 150, 200,300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900or 4000. The fragment length may be anywhere between said minimum andmaximum length.

In one preferred arrangement, the mutant HSV is HSV1716/CMV-NTR/GFPdeposited in the name of Crusade Laboratories Limited having an addressat Department of Neurology Southern General Hospital 1345 Govan RoadGovan Glasgow G51 5TF Scotland on 5 Nov. 2003 at the European Collectionof Cell Cultures (ECACC), Health Protection Agency, Porton Down,Salisbury, Wiltshire, SP4 0JG, United Kingdom under accession number03110501 in accordance with the provisions of the Budapest Treaty.

Suitably, the administration of said herpes simplex virus and/or saidNTR prodrug may comprise parenteral administration. Preferablyadministration of the herpes simplex virus is by injection, morepreferably injection to the tumour which is to be treated. The NTRprodrug may also be administered by injection, which may also comprisedirect injection to the site of the tumour. Alternatively injections maybe intravenous.

Administration of the herpes simplex virus and NTR prodrug may besimultaneous, e.g. by combining virus and prodrug in a singlecomposition, or be substantially simultaneous, e.g. one beingadministered immediately after the other. Alternatively, a predeterminedtime period may be provided between administration of the herpes simplexvirus and the NTR prodrug. The invention is not limited by the order ofadministration.

In a further aspect of the present invention in vitro or in vivo methodsare provided for delivery of nucleic acid encoding a nitroreductase toat least one cell or to a tissue of interest said method comprising thestep of infecting said cell(s) or tissue with a herpes simplex virusaccording to the invention.

In another aspect of the invention, a kit of parts is providedcomprising a first container in which a quantity of herpes simplex virusaccording to the invention is provided and a second container in which aquantity of NTR prodrug is provided. Instructions for theadministration, optionally including information on suitable dosages ofherpes simplex virus and/or the NTR prodrug, may also be provided withthe kit.

In another aspect of the present invention a method of making orproducing a modified herpes simplex virus of the invention is providedcomprising the step of introducing a nucleic acid sequence encoding anitroreductase at a selected and/or predetermined insertion site in thegenome of a selected herpes simplex virus.

As described, the nucleic acid sequence encoding the nitroreductase mayform part of a nucleic acid cassette which is inserted in the genome ofa selected herpes simplex virus by homologous recombination. Whetherpart of a cassette or not, the site of insertion may be in any genomiclocation selected. One preferred insertion site is in one or both of thelong repeat regions (R_(L)), and one copy of the cassette is preferablyinserted in each copy of the long repeat (R_(L)). More preferably theinsertion site is in at least one (preferably both) RL1 locus and mostpreferably it is inserted in at least one (preferably both) of theICP34.5 protein coding sequences of the HSV genomic DNA. It is preferredthat the insertion occurs in identical or substantially similarpositions in each of the two repeat regions, RL1 loci or ICP34.5 proteincoding sequences.

Insertion may be such as to produce a modified virus which is anon-neurovirulent mutant capable of expressing the encodednitroreductase polypeptide upon transfection into mammalian, morepreferably human, cells in vivo and in vitro in a form which isfunctional to facilitate the uptake and/or activation of NTR prodrug.The non-neurovirulent mutant may be an ICP34.5 null mutant.

The nucleic acid cassette may be of any size, e.g. up to 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 kbp in length.

Preferably, the herpes simplex virus contains at least one copy of thenucleic acid encoding the nitroreductase in each long repeat region(R_(L)), i.e. in the terminal and internal long repeat (TR_(L) andIR_(L)) regions. In a preferred arrangement each exogenous sequence orcassette is located in an RL1 locus of the herpes simplex virus genome,more preferably in the DNA of the herpes simplex virus genome encodingthe ICP34.5 gene or protein coding sequence. The herpes simplex virusthereby lacks neurovirulence.

The parent herpes simplex virus, from which a virus of the invention isderived may be of any kind, e.g. HSV-1 or HSV-2. In one preferredarrangement the herpes simplex virus is a variant of HSV-1 strain 17 andmay be obtained by modification of the strain 17 genomic DNA. Suitablemodifications include the insertion of the exogenous nitroreductasenucleic acid sequence or exogenous/heterologous cassette comprising saidsequence into the herpes simplex virus genomic DNA. The insertion may beperformed by homologous recombination of the exogenous nucleic acidsequence into the genome of the selected herpes simplex virus.

Although the non-neurovirulent phenotype of the herpes simplex virus ofthe invention may be the result of insertion of the exogenous nucleicacid sequence in the RL1 locus, herpes simplex viruses according to thepresent invention may be obtained by utilising a non-neurovirulentparent strain, e.g. HSV1716 deposited under the Budapest Treaty at theEuropean Collection of Animal Cell Cultures (ECACC), Health ProtectionAgency, Porton Down, Salisbury, Wiltshire, United Kingdom underaccession number V92012803, and inserting the exogenous nucleic acidsequence at another location of the genome by standard geneticengineering techniques, e.g. homologous recombination. In this aspectthe location of the herpes simplex virus genome selected for insertionof the nitroreductase nucleic acid sequence or cassette containing saidsequence may be a neutral location.

Herpes simplex viruses of the present invention may be variants of aknown ‘parent’ strain from which the herpes simplex virus of theinvention has been derived. A particularly preferred parent strain isHSV-1 strain 17. Other parent strains may include HSV-1 strain F orHSV-2 strain HG52. A variant comprises an HSV in which the genomesubstantially resembles that of the parent, contains the nitroreductasenucleic acid sequence or cassette containing said sequence and maycontain a limited number of other modifications, e.g. one, two or threeother specific mutations, which may be introduced to disable thepathogenic properties of the herpes simplex virus, for example amutation in the ribonucleotide reductase (RR) gene, the 65K transinducing factor (TIF) and/or a small number of mutations resulting fromnatural variation, which may be incorporated naturally duringreplication and selection in vitro or in vivo. Otherwise the genome ofthe variant will be that of the parent strain.

Herpes simplex viruses of the invention may be used alone, or incombination with an NTR prodrug in a method of medical treatment. Thismay involve treatment of diseases associated with or involving theproliferation of cells, or cancers or tumours of any kind. Treatment mayinvolve the selective lysis of dividing cells. This may be oncolysis,i.e. lysis of tumour cells. Tumours to be treated may be of any kind,may comprise cancers, neoplasms or neoplastic tissue and may be in anyanimal or human patient.

Cancer/tumour types which may be treated may be primary or secondary(metastatic) tumours. Tumours to be treated may be nervous systemtumours originating in the central or peripheral nervous system, e.g.glioma, medulloblastoma, meningioma, neurofibroma, ependymoma,Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma, or maybe non-nervous system tumours originating in non-nervous system tissuee.g. melanoma, mesothelioma, lymphoma, hepatoma, epidermoid carcinoma,prostate carcinoma, breast cancer cells, lung cancer cells or coloncancer cells. HSV mutants of the present invention may be used to treatmetastatic tumours of the central or peripheral nervous system whichoriginated in a non-nervous system tissue.

Herpes simplex viruses of the invention may be used in ‘gene delivery’methods in vitro or in vivo. Non-neurovirulent herpes simplex viruses ofthe invention are expression vectors and may be used to infect selectedcells or tissues in order to express the nitroreductase encoded by theherpes simplex virus genome.

In one arrangement, cells may be taken from a patient, a donor or fromany other source, infected with a herpes simplex virus of the invention,optionally screened for expression and/or function of the encodednitroreductase, and optionally returned/introduced to a patient's body,e.g. by injection.

Delivery of herpes simplex viruses of the invention to the selectedcells may be performed using naked virus or by encapsulation of thevirus in a carrier, e.g. nanoparticles, liposomes or other vesicles.

In vitro cultured cells, preferably human or mammalian cells,transformed with viruses of the present invention and preferably cellsexpressing the nitroreductase protein as well as methods of transformingsuch cells in vitro with said viruses form further aspects of thepresent invention.

In this specification, a mutant herpes simplex virus is a non-wild typeherpes simplex virus and may be a recombinant herpes simplex virus.Mutant herpes simplex viruses may comprise a genome containingmodifications relative to the wild type. A modification may include atleast one deletion, insertion, addition or substitution.

Medicaments and pharmaceutical compositions according to aspects of thepresent invention may be formulated for administration by a number ofroutes, including but not limited to, parenteral, intravenous,intramuscular, intratumoural, oral and nasal. The medicaments andcompositions may be formulated in fluid or solid (e.g. tablet) form.Fluid formulations may be formulated for administration by injection toa selected region of the human or animal body.

In this specification, non-neurovirulence is defined by the ability tointroduce a high titre of virus (approx 10⁶ plaque forming units (pfu))to an animal or patient^(22,23) without causing a lethal encephalitissuch that the LD₅₀ in animals, e.g. mice, or human patients is in theapproximate range of ≧10⁶ pfu²¹.

Where all copies of the ICP34.5 gene present in the herpes simplex virusgenome (two copies are normally present) are disrupted such that theherpes simplex virus is incapable of producing a functional ICP34.5 geneproduct, the virus is considered to be an ICP34.5 null mutant.

A regulatory sequence (e.g. promoter) that is operably linked to anucleotide sequence may be located adjacent to that sequence or in closeproximity such that the regulatory sequence can effect and/or controlexpression of a product of the nucleotide sequence. The encoded productof the nucleotide sequence may therefore be expressible from thatregulatory sequence.

NTR Prodrug

In this specification, “NTR prodrug” means any chemical compound oragent which is not toxic, or exhibits low toxicity, to a selected humanor animal body, or to selected cells or tissues of the human or animalbody, and which may be activated by a nitroreducase enzyme to a chemicalcompound or agent which is cytotoxic to the human or animal body or tothose selected cells.

“Activation” may involve conversion of a non-toxic (or low toxicity)prodrug to an active cytotoxic form. That conversion may involveenzymatic reduction of the prodrug by NTR. The enzymatic reductionreaction may involve the prodrug as a substrate for NTR and may involveother co-factors.

Examples of NTR prodrugs may include compounds from the followingclasses of molecules:

-   -   1. dinitirobenzamides;    -   2. dinitroaziridinylbenzamides (e.g. CB1954);    -   3. dinitrobenzamide mustard derivatives (e.g. SN23862);    -   4. 4-nitrobenzylcarbamates;    -   5. nitroindolines;    -   6. nitroaromatics that are substrates of NTR and are activated        to release a cytotoxic phosphoramide mustard or like-reactive        species upon NTR-reduction (also called nitroaryl        phosphoramides)¹⁷;    -   7. nitroaromatic prodrugs of the dinitrobenzamide class.        Examples of NTR prodrugs are disclosed in references 16 and 17        which are incorporated herein in their entirety by reference.

Nitroreductase (NTR)

Nitroreductase enzymes commonly catalyze the reduction of nitrocompounds, quinones, and dyes. The enzymatic reduction may involve theco-factor NADPH.

In this specification nitroreductase (NTR) refers to an enzyme capableof activating an NTR prodrug to an active cytotoxic form.

Preferred NTR's may have the ability to reduce a wide range ofnitro-containing compounds such as nitrofurazone (to the hydroxylamines)and quinones such as menadione (to the quinols).

Preferred NTR's may be specifically inhibited by the irreversibleinhibitor dicoumarol.

One preferred NTR is the E. coli oxygen insensitive nitroreductaseenzyme (the nfsB gene product). Sequence information for E. coli NTR canbe found at the NCBI database (http://www.ncbi.nlm.nih.gov/) (underaccession numbers BA000007 (GI:47118301)—E. coli complete genomesequence—and BAB34039 (GI:13360074)-nitroreductase sequence information.

The amino acid sequence for the E. coli NTR protein (SEQ ID No. 1) andpolynucleotide sequence for the E. coli NTR gene (SEQ ID No. 2) arereproduced at FIGS. 32(A) and (B) respectively.

The nucleotide and amino acid sequences of suitable nitroreductaseenzymes may be derived or obtained from any animal, insect ormicroorganism including humans, non-human mammals and bacteria and maybe selected from those sequences which are publicly available. Manysequences for other nitroreductase genes are publicly available.Examples of other nitroreductase nucleic acid sequences which may formpart of a herpes simplex virus according to the present inventioninclude the following which are referred to by their accession numberfor the NCBI database (www.ncbi.nlm.nih.gov):

-   -   BAB34039 (GI:13360074)—E. coli    -   BAA35218.1 (GI:1651240)—E. coli    -   AAB72053.1 (GI:2415385)—B. subtilis.

Hybridisation Stringency

In accordance with the present invention, nucleic acid sequences may beidentified by using hybridization and washing conditions of appropriatestringency.

Complementary nucleic acid sequences will hybridise to one anotherthrough Watson-Crick binding interactions. Sequences which are not 100%complementary may also hybridise but the strength of the hybridisationusually decreases with the decrease in complementarity. The strength ofhybridisation can therefore be used to distinguish the degree ofcomplementarity of sequences capable of binding to each other.

The “stringency” of a hybridization reaction can be readily determinedby a person skilled in the art.

The stringency of a given reaction may depend upon factors such as probelength, washing temperature, and salt concentration. Higher temperaturesare generally required for proper annealing of long probes, whileshorter probes may be annealed at lower temperatures. The higher thedegree of desired complementarity between the probe and hybridisablesequence, the higher the relative temperature which can be used. As aresult, it follows that higher relative temperatures would tend to makethe reaction conditions more stringent, while lower temperatures lessso.

For example, hybridizations may be performed, according to the method ofSambrook et al., (“Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1989) using a hybridization solutioncomprising: 5×SSC, 5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/mldenatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate andup to 50% formamide. Hybridization is carried out at 37-42° C. for atleast six hours. Following hybridization, filters are washed as follows:(1) 5 minutes at room temperature in 2×SSC and 1% SDS; (2) 15 minutes atroom temperature in 2×SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37° C.in 1×SSC and 1% SDS; (4) 2 hours at 42-65° C. in 1×SSC and 1% SDS,changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required toachieve hybridization between nucleic acid molecules is to calculate themelting temperature T_(m) (Sambrook et al., 1989):

T _(m)=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/n

where n is the number of bases in the oligonucleotide.

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the T_(m) is 57° C. The T_(m) of a DNA duplex decreases by 1-1.5°C. with every 1% decrease in sequence complementarity.

Accordingly, nucleotide sequences can be categorised by an ability tohybridise to a target sequence under different hybridisation and washingstringency conditions which can be selected by using the above equation.The T_(m) may be used to provide an indicator of the strength of thehybridisation.

The concept of distinguishing sequences based on the stringency of theconditions is well understood by the person skilled in the art and maybe readily applied.

Sequences exhibiting 95-100% sequence complementarity may be consideredto hybridise under very high stringency conditions, sequences exhibiting85-95% complementarity may be considered to hybridise under highstringency conditions, sequences exhibiting 70-85% complementarity maybe considered to hybridise under intermediate stringency conditions,sequences exhibiting 60-70% complementarity may be considered tohybridise under low stringency conditions and sequences exhibiting50-60%% complementarity may be considered to hybridise under very lowstringency conditions.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Generation of plasmid RL1.dIRES-GFP from plasmids pNAT-IRES-GFPand RL1.del.

FIG. 2. Agarose gel electrophoresis of HpaI digested, CIP treated,RL1.del. RL1.del was digested with HpaI. The digested DNA was thentreated with Calf Intestinal Phosphatase (CIP) to prevent the vectorre-annealing to itself in subsequent ligation reactions. A sample of thedigested/CIP treated DNA was electrophoresed, beside a 1 Kbp DNA ladder(Promega), on a 1% agarose gel. HpaI linearises the vector at 8.6 Kbp.

FIG. 3. Agarose gel electrophoresis of NsiI/SspI digested pNAT-IRES-GFP(A) and purified/blunt-ended pCMV-NAT-IRES-GFP-PolyA (B). Four NsiI/SspIdigestions of pNAT-IRES-GFP were electrophoresed, beside a 1 Kbp DNALadder (Promega) on a 1% agarose gel. The 5.4 Kbp fragments(pCMV-NAT-IRES-GFP-PolyA) were purified from the gel. The purified DNAwas blunt ended using Klenow polymerase and a sample electrophoresed onan agarose gel to check its concentration.

FIG. 4. Identification of RL1.del clones containing thepCMV-NAT-IRES-GFP-PolyA insert. Ligation reactions were set up with thepurified, blunt ended pCMV-NAT-IRES-GFP-PolyA fragment and HpaIdigested, CIP treated RL1.del. Bacteria were transformed with samplesfrom the ligation reactions and plated out onto LBA (Amp^(r)) plates.Colonies were picked and plasmid DNA was extracted and digested withAflII. Digested samples were electrophoresed, beside a 1 Kbp DNA ladder(L) (Promega), on a 1% agarose gel.

*Clones 5 and 8 contained the pCMV-NAT-IRES-GFP-PolyA insert as twofragments of the predicted size—4.8 Kbp and 9.2 Kbp—were generated fromAflII digestion. Clones without inserts would not be digested with AflIIas there is no AflII site in RL 1.del. N.B. Inserts could have beencloned in two orientations, both of which were acceptable.

FIG. 5. Determination of the orientation of pCMV-NAT-IRES-GFP-PolyA inclone 5 (RL1.dCMV-NAT-GFPb). pCMV-NAT-IRES-GFP-PolyA (blunt ended) couldhave been cloned into the HpaI site of RL1.del in two orientations. Todetermine the orientation of the insert in clone 5, the plasmid wasdigested with XhoI and the digested DNA electrophoresed, beside a 1 KbpDNA ladder (Promega), on a 1% agarose gel. If the insert had been clonedin the orientation shown in A, two fragments of 10.2 Kbp and 3.8 Kbpwould be generated from XhoI digestion. If it had been cloned in theopposite orientation (B), two fragments of 12.4 Kbp and 1.6 Kbp would begenerated. The presence of two fragments of 10.2 Kbp and 3.8 Kbp in thegel confirmed that the insert had been cloned in the orientation shownin A.

*This XhoI site was present in the initial cloning vector (RL1.del),upstream of the HpaI site into which pCMV-NAT-IRES-GFP-PolyA was cloned.

FIG. 6. Removal of pCMV-NAT from clone 5 (A) and large scale plasmidpreparation of RL1.dIRES-GFP (B). Four samples of clone 5 were digestedwith XhoI and electrophoresed, beside a 1 Kbp DNA ladder (L) (Promega),on a 1% agarose gel (A). The larger fragment of DNA generated from thisdigestion (10.2 Kbp) was purified from the gel and ligated backtogether, at the XhoI sites, to form a single XhoI site in a newplasmid, designated RL1.dIRES-GFP. A large-scale plasmid preparation wasgrown up and the preparation checked by digesting with XhoI. 1 μl and 4μl of the digested DNA was electrophoresed, beside a 1 Kbp DNA ladder(L) (Promega), on a 1% agarose gel (B). The DNA should produce a singlefragment of 10.2 Kbp when digested with XhoI. The ClaI, BglII, NruI andXhoI sites of RL1 dIRES-GFP are all unique.

*Clone 5 is the RL1.del plasmid into which has been cloned the 5.4 KbppCMV-NAT-IRES-GFP-PolyA fragment from pNAT-IRES-GFP.

FIG. 7. Generation, detection and purification of ICP34.5 null HSV-1expressing a gene product of interest.

FIG. 8. Strategy used to clone pCMV-NTR from pPS949 into RL1.dIRES-GFP.(1) Digest pPS949 with BamHI and purify the 1.6 Kbp pCMV-NTR fragment;(2) Digest RL1 dIRES-GFP with BglII and treat with Calf IntestinalPhosphatase (CIP); (3) Clone the pCMV-NTR fragment (BamHI ends) into theBglII site of RL1.dIRES-GFP.

*The pPS949 plasmid was a kind gift from Professor Lawrence Young(University of Birmingham) and contains the E. coli nitroreductase (NTR)gene downstream of the CMV-IE promoter (pCMV) in pLNCX (Clontech).

FIG. 9. Agarose gel electrophoresis of BamHI digested pPS949 (A) and thepurified pCMV-NTR fragment (B). Four samples of pPS949 were digestedwith BamHI and electrophoresed, beside a 1 Kbp DNA ladder (L) (NewEngland Biolabs), on a 1% agarose gel. The 1.6 Kbp fragments, consistingof the E. coli nitroreductase (NTR) gene downstream of the CMV IEpromoter (pCMV), were purified from the gel and a sample of the purifiedDNA was electrophoresed on an agarose gel to check its concentration.

FIG. 10. Agarose gel electrophoresis of BglII digested, CIP treatedRL1.dIRES-GFP.RL1.dIRES.GFP was digested with BglII. The digestedplasmid was then treated with Calf Intestinal Phosphatase (CIP) toprevent the vector re-annealing to itself in subsequent ligationreactions. A sample of the digested/CIP treated DNA was electrophoresed,beside a 1 Kbp DNA ladder (Promega), on a 1% agarose gel to check itsconcentration. pCMV-NTR from pPS949 was subsequently cloned into thisdigested/CIP treated vector.

FIG. 11. Determination of the orientation of pCMV-NTR in clone 4.pCMV-NTR (BamHI ends) could have been cloned into the BglII site ofRL1.dIRES-GFP in two orientations. To determine the orientation, clone 4was digested with BglII and XhoI and the digested DNA electrophoresed,beside a 1 Kbp DNA ladder (Promega), on a 1% agarose gel. If the insertwas in the desired orientation (A), two fragments (11.5 Kbp and 300 bp)would be generated. If in the opposite orientation, two fragments of10.5 Kbp and 1.3 Kbp would be generated. The presence of a band at 300bp (and the absence of a band at 1.3 Kbp) confirmed that the pCMV-NTRfragment had been cloned into the vector in the desired orientation.

FIG. 12. Agarose gel electrophoresis of ScaI digested clone 4 (A) andHSV1716/CMV-NTR/GFP viral titres (B). Clone 4 (RL1.dCMV-NTR-GFP) wasdigested with ScaI, the digested DNA purified and 5 μl electrophoresed,beside a 1 Kbp DNA ladder (Promega), on a 1% agarose gel, to check itsconcentration. 80% confluent BHK cells were then co-transfected with 10μl HSV17⁺ DNA and an appropriate volume of the remaining digested clone4. The cells were incubated at 37° C. for 3 days until cpe was evident.Recombinant viral plaques were picked under the fluorescent microscope,purified and a virus stock, named HSV1716/CMV-NTR/GFP, grown up. Thecell-associated and cell-released fraction of the virus stock wastitrated on BHK cells.

FIG. 13. Growth kinetics of HSV17, HSV1716 and HSV1716/CMV-NTR/GFP inconfluent BHK and 3T6 cells. Confluent BHK and 3T6 cells were infectedat a MOI of 0.1 pfu/cell. Infected cells were harvested at 0, 4, 24, 48and 72 hrs post infection, sonicated and progeny virus titrated on BHKcell monolayers. All viruses replicated with similar kinetics in BHKcells (A); HSV1716 and HSV1716/CMV-NTR/GFP both failed to replicateefficiently in confluent 3T6 cells (B).

FIG. 14. Western blot analysis of ICP34.5 expression in HSV17⁺ andHSV1716/CMV-NTR/GFP infected BHK cells. BHK cells were infected withHSV17⁺ and HSV1716/CMV-NTR/GFP at a MOI of 10 pfu/cell. 16 hrs postinfection, the cells were harvested and protein extracts analysed using10% SDS-PAGE in a Western blot using a polyclonal anti-ICP34.5 antibody.ICP34.5 was strongly expressed in HSV17⁺ infected cells but was notexpressed in HSV1716/CMV-NTR/GFP infected cells.

FIG. 15. Western blot analysis of NTR expression in HSV1716/CMV-NTR/GFPinfected cell lines. BHK, C8161, VM and 3T6 cells were infected with 10pfu/cell HSV1716/CMV-NTR/GFP, HSV17 or mock infected. 16 hrs postinfection, the cells were harvested and protein extracts analysed in aWestern blot using a polyclonal NTR-specific antibody. Significant NTRexpression was detected in all the HSV1716/CMV-NTR/GFP infected cells.No NTR expression was detected in the mock or HSV17⁺ infected cells.

FIG. 16. Effect of HSV1716/CMV-NTR/GFP and HSV1716-GFP with or withoutCB1954 (50M) on confluent 3T6 cells. Confluent 3T6 cells in three wellsof a 96-well plate were mock infected, infected with 1 or 10 pfu/cellHSV1716/CMV-NTR/GFP or infected with 1 pfu/cell of HSV1716-GFP. 45minutes later, infected cells were overlaid with media containing 50MCB1954 or with media alone and incubated at 37° C. 24, 48, 72, 96, and120 hrs later, % cell survival was determined relative to that of mockinfected cells without prodrug using CellTiter 96 Aqueous One SolutionCell Proliferation Assay (Promega). Figures shown represent the mean of3 values+/−standard error of the mean.

FIG. 17. Effect of HSV1716/CMV-NTR/GFP and HSV1716-GFP with or withoutCB1954 (50M) on confluent C8161 cells. Confluent C8161 cells in threewells of a 96-well plate were mock infected, infected with 1 or 10pfu/cell HSV1716/CMV-NTR/GFP or infected with 1 pfu/cell of HSV1716-GFP.45 minutes later, infected cells were overlaid with media containing 50MCB1954 or with media alone and incubated at 37° C. 24, 48 and 72 hrslater, % cell survival was determined relative to that of mock infectedcells without prodrug using CellTiter 96 Aqueous One Solution CellProliferation Assay (Promega). Figures shown represent the mean of 3values+/−standard error of the mean.

FIG. 18. Confluent 3T6 cells 72 hrs post treatment with 10 pfu/cellHSV1716/CMV-NTR/GFP (A), or 10 pfu/cell HSV1716/CMV-NTR/GFP with 50 μMCB1954 (B). The extent of cell death is significantly more pronounced inHSV1716/CMV-NTR/GFP infected cells overlaid with media containing 50MCB1954 than in HSV1716/CMV-NTR/GFP infected cells overlaid with normalmedia. The extent of cell death following infection of these cells with10 pfu/cell HSV1716, with or without CB1954, is comparable to that seenin A (data not shown). 50M CB1954 alone has no effect on these cells.

FIG. 19. Confluent C8161 cells 72 hrs post treatment with 10 pfu/cellHSV1716/CMV-NTR/GFP (A), or 10 pfu/cell HSV1716/CMV-NTR/GFP with 50 μMCB1954 (B). The extent of cell death is significantly more pronounced inHSV1716/CMV-NTR/GFP infected cells overlaid with media containing 50 μMCB1954 than in HSV1716/CMV-NTR/GFP infected cells overlaid with normalmedia. The extent of cell death following infection of these cells with10 pfu/cell HSV1716, with or without CB1954, is comparable to that seenin A (data not shown). 50 μM CB1954 alone has no effect on these cells.

FIG. 20. Weight change (as a guide to health) in athymic nude mice withsubcutaneous A2780 (xenograft) tumours injected intratumourally withHSV1790. Group size=3 mice per dose. A2780 xenografts at date ofintratumoural injection (Day 0) are between 0.5-1 mm in diameter. Thexenografts have reached this size 12 days after injection with 10million A2780 cells subcutaneously on the flank of female athymic nudemice.

FIG. 21. Change in tumour volume over time in athymic nude mice withA2780 xenografts after intratumoural injection of HSV1790.

FIG. 22. Starting tumour sizes of mice.

FIG. 23. Alterations in weight after treatment with CMV-ntr, CB1954 or acombination of both.

FIG. 24. Change in tumour volume after treatment with CMV-ntr, CB 1954or a combination of both.

FIG. 25. Starting tumour volume of each treatment group (see Table 2).

FIG. 26. Weight (as a measurement of health) in athymic nude mice withA2780 xenograft treated with either HSV1790, HSV1716, CB1954 or acombination of them.

FIG. 27. Change in tumour volume of xenografts treated with the prodrugCB1954.

FIG. 28. Changes in tumour volume in xenograft treated with 10⁵ PFUHSV1790 and CB1954.

FIG. 29. Changes in tumour volume in xenografts treated with 10⁶ PFUHSV1790 and CB1954.

FIG. 30. Changes in tumour volume in xenografts treated with 10⁵ PFUHSV1716 and CB1954.

FIG. 31. Comparison of 10⁵ PFU, 10⁶ PFU HSV1790 and 10⁵ PFU HSV1716.

FIG. 32. Sequence information for E. coli NTR. (A) Amino acid sequenceof NTR polypeptide (SEQ ID No. 1); (B) polynucleotide sequence for NTRgene (SEQ ID No.2).

FIG. 33. Structure of two NTR prodrugs. (A) CB1954; (B) SN23862.

DETAILED DESCRIPTION OF THE BEST MODE OF THE INVENTION

Specific details of the best mode contemplated by the inventors forcarrying out the invention are set forth below, by way of example. Itwill be apparent to one skilled in the art that the present inventionmay be practiced without limitation to these specific details.

Vectors Useful for Generation of Herpes Simplex Virus Mutants

Mutant herpes simplex viruses of the invention may be generated by useof nucleic acid vectors.

One such vector useful for generation of mutant herpes simplex virusesaccording to the present invention is a nucleic acid vector comprising,consisting or consisting essentially of:

first and second nucleotide sequences corresponding to nucleotidesequences flanking an insertion site in the genome of a selected herpessimplex virus; anda cassette located between said first and second nucleotide sequencescomprising nucleic acid encoding:

a) one or a plurality of insertion sites; and

b) a ribosome binding site; and

c) a marker.

Another vector useful for generation of mutant herpes simplex virusesaccording to the present invention is a nucleic acid vector comprising,consisting or consisting essentially of:

first and second nucleotide sequences corresponding to nucleotidesequences flanking an insertion site in the genome of a selected herpessimplex virus; anda cassette located between said first and second nucleotide sequencescomprising nucleic acid encoding:

a) one or a plurality of insertion sites; and

b) a first regulatory nucleotide sequence; and

c) a marker.

The first and second nucleotide sequences may correspond to nucleotidesequences flanking an insertion site formed in, or comprising all or apart of, the ICP34.5 protein coding sequence of the genome of a selectedherpes simplex virus.

The cassette may comprise a plurality of insertion sites, each insertionsite preferably formed by nucleic acid encoding a specific restrictionendonuclease site (‘restriction site’). Together the restriction sitesmay form a multiple cloning site (MCS) comprising a series ofoverlapping or distinct restriction sites, preferably a series ofdistinct restriction sites comprising one or more of the ClaI, BglII,NruI, XhoI restriction sites.

The encoded components of the cassette may be arranged in apredetermined order. In one arrangement, the one or plurality ofinsertion sites is/are arranged upstream (i.e. 5′) of the ribosomebinding site/first regulatory sequence and the ribosome bindingsite/first regulatory sequence is arranged upstream (i.e. 5′) of themarker.

The first and second nucleotide sequences may comprise nucleotidesequences having identity to regions of the genome surrounding theinsertion site in the selected herpes simplex virus (the ‘viralinsertion site’). These sequences enable the cassette to be incorporatedat the viral insertion site by homologous recombination between thefirst and second nucleotide sequences and their respective correspondingsequences in the viral genome.

Thus the first and second nucleotide sequences are flanking sequencesfor homologous recombination with corresponding sequences of a selectedviral genome, such homologous recombination resulting in insertion ofthe cassette at the viral insertion site.

The first and second nucleotide sequences may correspond to nucleotidesequences flanking an insertion site in the RL1 locus of the HSV genome,more preferably in the ICP34.5 protein coding sequence of the HSVgenome.

The first and second nucleotide sequences may each be at least 50 bp inlength, more preferably at least 100, 150, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 19002000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100,3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900 or 4000 bp in length.Each of the first and second nucleotide sequences may have at least 50%sequence identity to their corresponding sequence in the viral genome,more preferably at least 60%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%,96%, 97%, 98% 99% or 100% identity. Identity of sequences is determinedacross the entire length of a given nucleotide sequence. Where sequencesare of different length, sequence identity of the shorter sequence isdetermined over the entire length of the longer sequence.

The first and second nucleotide sequences may be characterised by theability of one strand of a given sequence to hybridise with thecorresponding single-stranded complement of the HSV genome under varyinghybridisation stringency conditions. Suitably, the first and secondnucleotide sequences will hybridise with their corresponding complementunder very low, low or intermediate stringency conditions, morepreferably at high or very high stringency conditions.

The viral insertion site is the position between the genomic nucleotidesequences corresponding to the first and second nucleotide sequences ofthe vector (the ‘genomic’ and ‘vector flanking sequences’ respectively)at which homologous recombination will occur and may be predetermined byselection of the vector flanking sequences. Where the genomic flankingsequences are immediately adjacent, the insertion site is the positionbetween the peripheral and immediately adjacent bases of the two genomicflanking sequences, such that insertion of the cassette separates thegenomic flanking sequences. Where the genomic flanking sequences areseparated by one or a plurality of bases in the viral genome, theinsertion site is formed by said one or a plurality of bases which areexcised from the genome by the homologous recombination event.

The position of the viral insertion site may be accurately selected bycareful selection and construction of the vector flanking sequences.Accordingly, the vector may be constructed such that homologousinsertion of the cassette results in disruption of a chosen proteincoding sequence and inactivation of the respective gene product or suchthat the cassette is inserted at a non-protein coding region of theviral genome. The complete genome sequences of several herpes simplexvirus strains have been reported and are publicly available. Thecomplete genome sequence for HSV-1 strain 17syn+ was reported by Dolanet al³ (incorporated herein by reference) and the complete genomesequence of HSV-2 strain HG52 was reported by Dolan et al⁴ (incorporatedherein by reference) and is available from the EMBL database underaccession code Z86099. Using this information, the vector of the presentinvention may preferably be designed for use in generating mutant HSV-1(e.g. in strain 17 or F) or mutant HSV-2 (e.g. in strain HG52).

The first and second nucleotide sequences (vector flanking sequences)may each comprise sequence corresponding to the RL terminal repeatregion of the genome of the selected HSV (e.g. HSV-1 strains 17 or F orHSV-2 strain HG52). The vector flanking sequences may comprise, consistor consist essentially of nucleotide sequences of the RL repeat regionwhich flank the ICP34.5 protein coding sequence. In flanking the ICP34.5coding sequence, one or both of the selected sequences may, in thecorresponding HSV genome, overlap, i.e. extend into, the ICP34.5 proteincoding sequence or one or both sequences may be selected so as to notoverlap the ICP34.5 protein coding sequence. In a similar manner, theselected sequences may be chosen to overlap completely or partiallyother important encoded signals, e.g. transcription initiation site,polyadenylation site, defined promoters or enhancers. In this preferredarrangement the insertion site will thus comprise all or a part of theICP34.5 protein coding sequence and/or be such that the insertedcassette disrupts the ICP34.5 protein coding sequence.

The vectors described, comprising first and second nucleotide sequencescorresponding to regions of the RL repeat region flanking and/oroverlapping the ICP34.5 protein coding sequence, may be used in thegeneration of ICP34.5 null mutants wherein all or a portion of theICP34.5 protein coding sequence is excised and replaced during thehomologous recombination event such that both copies of the ICP34.5coding sequence are disrupted.

The recombination may result in an insertion of nucleic acid within theICP34.5 protein coding sequence thereby disrupting that sequence. Inthat case, successfully transformed virus are thus mutants incapable ofgenerating the ICP34.5 active gene product from at least one copy, andpreferably from both copies, of the ICP34.5 gene.

Successfully transformed viruses are thus mutants incapable ofgenerating the ICP34.5 active gene product.

Each component of the cassette may be positioned substantially adjacentthe neighbouring component such that a single bicistronic transcriptcomprising or consisting essentially of the mRNA encoding the nucleotidesequence of interest, ribosome binding site and marker is obtainable.

The vectors described may further comprise, consist, or consistessentially of a nucleic acid encoding a selectable marker such as apolypeptide or protein conferring antibiotic resistance e.g. kanamycinresistance or ampicillin resistance.

The vectors described are preferably DNA vectors, particularly dsDNAvectors. The vector may be provided as a linear or circular (plasmid)DNA vector. The vector preferably contains nucleotide sequences, e.g.restriction endonuclease site(s), permitting transition between the twoforms by use of DNA ligation and restriction materials (e.g. enzymes)and techniques known to the person skilled in the art. To achievehomologous recombination with a selected HSV, the vector is preferablyprovided in linear form.

One such vector provided by the inventors is plasmid RL1.dIRES-GFPdeposited in the name of Crusade Laboratories Limited having an addressat Department of Neurology Southern General Hospital 1345 Govan RoadGovan Glasgow G51 5TF Scotland on 3 Sep. 2003 at the European Collectionof Cell Cultures (ECACC), Health Protection Agency, Porton Down,Salisbury, Wiltshire, SP4 0JG, United Kingdom under accession number03090303 in accordance with the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure (herein referred to as the ‘BudapestTreaty’).

RL1.dIRES-GFP provides a platform for generating a plurality of ‘shuttlevectors’ which can exploit the process of homologous recombination totransfer a nucleotide sequence of interest (downstream of a selectedpromoter) into the disabling RL1 locus of HSV-1, generating easilyidentifiable, oncolytic, ICP34.5 null HSV-1 mutants expressing theproducts of the nucleotide sequence of interest, e.g. an RNA transcriptor a polypeptide, and GFP. RL1.dIRES-GFP thus provides for ease ofgeneration and purification of ICP34.5 null HSV.

RL1.dIRES-GFP is a useful vector for making second-generation oncolyticviruses having enhanced cytotoxic potential and which may express theproduct(s) of selected gene(s) to enhance the oncolytic and/ortherapeutic effect of the administered virus.

The RL1.dIRES-GFP plasmid incorporates a multi-cloning sequence (MCS),upstream of an internal ribosome entry site (IRES), the GFP gene andSV40 polyadenylation sequences flanked by HSV-1 RL1 sequences.Incorporation of the encephalomyocarditis virus IRES (EMCV IRES) permitstranslation of two open reading frames from a single transcribed mRNA.

Following generation of a specific shuttle vector by cloning of thenucleotide sequence of interest (and the selected promoter) intoRL1.dIRES-GFP, recombinant HSV-1 expressing the desired nucleic acidtranscript or protein, can be generated and purified within 2 weeks.This compares with 2-3 months using prior art protocols.

In the ICP34.5 null HSV generated using the RL1.dIRES-GFP plasmidprovided by the inventors transcription of both the nucleotide sequenceof interest and GFP as a single transcript is controlled by the samepromoter upstream of the nucleotide sequence of interest, thetranscribed IRES directing cap-independent translation of GFP. Thegenerated ICP34.5 null HSV are non-neurovirulent. By modifying theRL1.dIRES-GFP plasmid to incorporate appropriate flanking sequencessurrounding the cassette other gene-specific HSV null mutants expressingGFP can be generated.

RL1.dIRES-GFP is promoterless, thus enabling a promoter of choice to beincorporated in the homologously recombined shuttle vector forcontrolling expression of the nucleotide sequence of interest from theinserted cassette.

Plasmid RL1.dIRES-GFP or modified plasmid shuttle vectors thereoffurther comprising nucleotide sequence encoding a nucleic acidtranscript or polypeptide of interest may be provided in isolated orpurified form.

The vector may be a variant of plasmid RL1.dIRES-GFP.

As the plasmid RL1.dIRES-GFP is designed for tandem expression of asequence of interest and the marker gene encoding green fluorescentprotein (GFP). The sequence of interest is cloned into RL1.dIRES-GFPalong with its promoter (e.g. CMV) such that the promoter drivestranscription of an mRNA for the sequence of interest along with theIRES-GFP. Translation results in expression of the GFP from the internalribosomal entry site and the gene of interest and promoter must becloned into RL1.dIRES-GFP in the correct orientation to achieve this.There are a number of instances where this tandem expression arrangementmay be unsuitable and a variation of the cassette design is favourable.

One example is the expression of siRNAs as short hairpin RNAs using RNApolIII promoters such as H1 or U6. These promoters are unable to drivethe additional tandem expression of the IRES-GFP as the RNApolIIIexpression cassette is designed only to produce short transcripts.Additionally, sequences of interest derived from genomic DNA with strongmRNA shut-off signals in their 3′ untranslated regions may not supportIRES-GFP expression.

Thus in some cases a cassette may be provided in which the sequence ofinterest and marker are expressed separately from independent promoters.

One variant contains a cassette in which the ribosome binding site ofplasmid RL1.dIRES-GFP is replaced with a regulatory nucleotide sequence,preferably a strong, constitutive promoter such as thePhosphoglycerokinase promoter. The marker is thereby expressed under thecontrol of this (the ‘first’) regulatory sequence. The nucleotidesequence of interest (e.g. NTR, an antisense or siRNA) is expressedunder the control of a second regulatory sequence upstream (5′) of thenucleotide sequence of interest, e.g. the CMV promoter. This vectorvariant is particularly suitable for expression of siRNA where a weakpromoter may be used for expression of the siRNA molecule or where thenucleic acid encoding the NTR may have a strong termination signalmaking it difficult to transcribe or translate a single bi- orpoly-cistronic transcript encoding the NTR and marker sequence. In thisarrangement the transformed virus containing the cassette integrated inthe viral genome produces two separate transcripts under the control ofthe first and second promoters.

One such cassette was constructed in the following manner. The 1.3 kbpblunt-ended EcoRI/AfIII fragment that contains the PGK promoter/GFP genewas obtained by restriction digestion followed by Klenow treatment fromthe vector pSNRG and cloned into the RL1-del vector cut with therestriction enzyme NruI that generates blunt ends. Successful insertionof the PGK/GFP DNA was confirmed by BamHI digestion and the orientationof the inserted DNA identified using the unique XhoI site in RL1-del andthe BsrGI site at the 3′ end of PGK/GFP. Plasmids with PGK/GFP in bothforward and reverse orientation were obtained and the plasmids weredesignated RL1-dPGK/GFP for and RL1-dPGK/GFPrev. Expression of GFP wasconfirmed in BHK cells transfected with the forward and reverseorientation plasmids.

Thus, sequences of interest along with their own promoters (although itis preferred that the PGK promoter is not also used for this purpose)can then be cloned into either RL1-dPGK/GFP for or RL1-dPGK/GFPrev ineither orientation using the remaining unique BglII, XhoI or HpaI uniquerestriction enzyme sites. The resulting plasmid can be used to deriverecombinant HSV in which the marker GFP gene and the gene of interestare expressed independently from their own promoters.

The vectors described may be constructed for use in generatingengineered HSV-1 or HSV-2 by insertion of a nucleic acid cassettethrough a mechanism of homologous recombination between nucleotidesequences flanking the cassette and corresponding sequences in theselected herpes simplex virus genome.

The vectors described may comprise and have use as:

-   -   i) gene delivery (gene therapy) vectors for delivery of a        selected nucleotide sequence, e.g. NTR, to a specific locus of        the HSV genome; and/or    -   ii) expression vectors for expression of the delivered        nucleotide sequence of i) from the HSV genome under the control        of a selected regulatory element; and/or    -   iii) vectors for the generation of HSV gene-specific null        mutants wherein the cassette is inserted at a selected genomic        location to disrupt the protein coding sequence of a selected        HSV gene such that the gene product is inactive in the resultant        mutant virus.

The vectors described may be used in the manufacture of engineered genespecific HSV null mutants, i.e. HSV mutants incapable of expressing anactive gene product of a selected gene. They may be used in themanufacture of engineered viruses which express a selected protein fromonly one gene copy the other gene copy being disrupted or modified suchthat it cannot express a functional gene product. Such vectors may alsobe used in the manufacture of a medicament, preferably comprising saidgene specific HSV null mutant, for use in treating cancer and tumours,preferably by the oncolytic treatment of the tumour.

The vectors described may also be used in the manufacture of engineeredHSV mutants wherein the genome of the mutant HSV comprises an exogenousor heterologous gene which may have been inserted in the HSV genome byhomologous recombination of the cassette. Preferably, theexogenous/heterologous gene is expressed in the mutant HSV, whichexpression may be regulated by a regulatory element, e.g. promoter,forming part of the inserted cassette. Such vectors may be used in themanufacture of a medicament, preferably comprising the engineered HSVmutant, for use in the treatment of disease, including the oncolytictreatment of tumours.

The vectors described may also be used in the manufacture of anengineered HSV mutant wherein the genome of the mutant HSV comprises anexogenous/heterologous gene (i.e. a non-HSV originating gene) which mayhave been inserted in a protein coding sequence of the HSV genome byhomologous recombination of the cassette such that the mutant HSV isincapable of expressing the active gene encoded by said protein codingsequence and wherein the exogenous/heterologous gene product isexpressed under the control of a regulatory element. Preferably, theregulatory element forms part of the cassette. Such vectors may be usedin the manufacture of a medicament, preferably comprising the engineeredHSV mutant, for use in the treatment of disease, including the oncolytictreatment of tumours.

The vectors described may also be used in the manufacture of anengineered HSV mutant wherein the genome of the mutant HSV comprises anucleotide sequence which has been inserted in a protein coding sequenceof the HSV genome by homologous recombination of the cassette such thatthe mutant HSV is incapable of expressing the active gene encoded bysaid protein coding sequence and wherein the inserted nucleotidesequence is expressed under the control of a regulatory element toproduce a desired transcript. Preferably, the regulatory element formspart of the cassette. Such vectors may be used in the manufacture of amedicament, preferably comprising the engineered HSV mutant, for use inthe treatment of disease, including the oncolytic treatment of tumours.

The vectors described may be used to generate mutant HSV by insertingthe cassette into the genome of a selected HSV, the method of generationmay comprise providing a vector described above, where the vector is aplasmid, linearising the vector; and co-transfecting a cell culture withthe linearised vector and genomic DNA from said HSV.

The co-transfection may be carried out under conditions effective forhomologous recombination of said cassette into an insertion site of theviral genome.

The method may further comprise one or more of the steps of:

-   -   1) screening said co-transfected cell culture to detect mutant        HSV expressing said marker; and/or    -   2) isolating said mutant HSV; and/or    -   3) screening said mutant HSV for expression of the nucleotide        sequence of interest or the RNA or polypeptide thereby encoded;        and/or    -   4) screening said mutant HSV for lack of an active gene product;        and/or    -   5) testing the oncolytic ability of said mutant HSV to kill        tumour cells in vitro.

Example 1 Construction of Plasmid RL1.dIRES-GFP General Approach

Plasmid RL1.dIRES-GFP was generated in three stages, illustrated in FIG.1.

1. The DNA sequences containing the CMV IE promoter (pCMV), the NATgene, the internal ribosome entry site (IRES), the GFP reporter gene andthe SV40 polyadenylation sequences were excised from pNAT-IRES-GFP usingNsiI and SspI and purified.

2. The purified pCMV-NAT-IRES-GFP-PolyA DNA fragment was cloned intoRL1.del to form a new plasmid designated RL1.dCMV-NAT-GFP.

3. The pCMV-NAT DNA sequences of RL1.dCMV-NAT-GFP were excised usingXhoI and the remainder of the plasmid re-ligated to form a novel plasmiddesignated RL1.dIRES-GFP. This novel plasmid contained a multi-cloningsite (all sites shown are unique) upstream of an IRES, the GFP gene andthe SV40 polyA sequences all within the HSV-1 RL1 flanking sequences.Recombinant ICP34.5 null HSV-1, expressing a gene of interest in the RL1locus, can be generated by cloning the gene of interest (downstream of asuitable promoter) into the multi-cloning site and co-transfecting BHKcells with the plasmid and HSV-1 DNA. Recombinant virus expressing thetarget gene can be identified using GFP fluorescence.

Removal of the CMV promoter and noradrenaline transporter gene(pCMV-NAT) from RL1.dCMV-NAT-GFP, followed by re-ligation of theremainder of the plasmid, resulted in a novel plasmid (RL1.dIRES-GFP)containing a multi-cloning site (MCS), upstream of theencephalomyocarditis virus internal ribosome entry site (EMCV IRES), theGFP reporter gene and the SV40 PolyA sequences, all within RL1 flankingsequences. This novel arrangement of DNA sequences or ‘smart cassette’allows ICP34.5 null HSV-1, expressing a gene of interest in the RL1locus, to be easily generated by simply inserting the desired transgene(downstream of a suitable promoter) into the MCS and co-transfecting BHKcells with the plasmid and HSV-1 DNA. The IRES situated between the GFPgene and the MCS permits expression of two genes from the same promoterand so recombinant virus expressing the gene of interest also expressesGFP and can therefore be easily identified under a fluorescencemicroscope and purified.

Materials and Methods

1 μg of RL1 del* was digested with 10 units HpaI (Promega) in a suitablevolume of 10× buffer (Promega) and nuclease free water (Promega) at 37°C. for 16 hrs. The digested plasmid was then purified using the QIAquickPCR purification kit (Qiagen), treated with 10 units of Calf IntestinalPhosphatase (Promega), in a suitable volume of 10×CIP buffer andnuclease free water for 4 hrs at 37° C., before being purified againusing a Qiaquick PCR purification kit. 5 μl of the purified DNA waselectrophoresed on a 1% agarose gel to check its concentration (FIG. 2).

4×1 g of pNAT-IRES-GFP** was digested with 10 units of NsiI and 10 unitsof SspI in a suitable volume of 10× buffer (Promega) and nuclease freewater (Promega) at 37° C. for 16 hrs. The reaction mixture waselectrophoresed in a 1% agarose gel for 1 hr at 110 volts. The 5.4 KbpDNA fragment consisting of the CMV IE promoter (pCMV), upstream of thenoradrenaline transporter gene (NAT), the encephalomyocarditis virusinternal ribosome entry site (IRES), the gene for green fluorescentprotein (GFP) and the SV40 polyadenylation sequences (SV40 Poly A), wasexcised using a sterile scalpel and the DNA purified from the gel usinga QIAquick Gel Extraction kit (Qiagen). The eluted DNA was blunt endedusing 3 units Klenow Polymerase (Promega) in accordance with themanufacturers instructions and the DNA purified using a QIAquick PCRpurification kit (Qiagen). 5 μl of the purified DNA fragment waselectrophoresed on a 1% agarose gel to check its concentration (FIG. 3).

Ligation reactions were carried out in small eppendorf tubes containing5 units T4 DNA Ligase (Promega), a suitable volume of 10×DNA LigaseBuffer (Promega), nuclease free water (Promega) and various volumes ofthe HpaI digested/CIP treated RL1.del and blunt endedpCMV-NAT-IRES-GFP-SV40 Poly A DNA, at 16° C. overnight. Competent JM109bacterial cells (Promega) were then transformed with various aliqouts ofthe ligation reactions***. Colonies formed on the plates were picked,had their plasmid DNA extracted using a Qiagen Plasmid Mini kit andscreened for inserts using AflII (New England Biolabs) restrictionenzyme analysis. Plasmid DNA containing the insert would produce twofragments of 4.8 Kbp and 9.2 Kbp following digestion with AflII. Twoclones (clone 5 and 8) contained the insert (FIG. 4). The orientation ofthe insert in clone 5 (RL1.dCMV-NAT-GFP) was determined using XhoIrestriction enzyme analysis (FIG. 5).

To generate RL1.dIRES-GFP from clone 5, the CMV-NAT portion of theCMV-NAT-IRES-GFP-SV40 PolyA insert was removed by digesting 4×500 ng ofclone 5 with 10 units of XhoI in a suitable volume of buffer and water(Promega), overnight at 37° C. The digested DNA was electrophoresed on a1% agarose gel at 110 volts for 1 hr (FIG. 6A). The 10.2 Kbp fragmentconsisting of the IRES, the GFP gene, the SV40 PolyA sequences and RL1flanking sequences in a pGEM3Zf(−) (Promega) backbone, was excised usinga sterile scalpel and the DNA purified from the gel using a QIAquick GelExtraction kit.

Ligation reactions were performed in small eppendorf tubes containing100 ng-500 ng purified DNA, 3 units T4 DNA Ligase (Promega), a suitablevolume of 10×DNA Ligase Buffer (Promega) and nuclease free water(Promega) overnight at 16° C. Competent JM109 bacterial cells (Promega)were then transformed with various aliquots of the ligationreactions***. Colonies formed on the plates were picked, had theirplasmid DNA extracted using a Qiagen Plasmid Mini kit and screened usingXhoI (Promega) restriction enzyme analysis. Colonies containing plasmidDNA from which CMV-NAT had been removed would produce one fragment of10.2 Kbp when digested with XhoI. Several positive clones were found,one was isolated, and a large-scale plasmid preparation undertaken usingPromega's Wizard Plus Maxipreps kit. The large-scale plasmid preparationwas checked by digesting with XhoI (FIG. 6B). This plasmid DNA wassubsequently named ‘RL1.dIRES-GFP’.

Plasmid RL1.dIRES-GFP has been deposited in the name of CrusadeLaboratories Limited having an address at Department of NeurologySouthern General Hospital 1345 Govan Road Govan Glasgow G51 5TF Scotlandon 3 Sep. 2003 at the European Collection of Cell Cultures (ECACC),Health Protection Agency, Porton Down, Salisbury, Wiltshire, SP4 0JG,United Kingdom under accession number 03090303 in accordance with theprovisions of the Budapest Treaty.

RL1.del

*RL1.del was provided by Dr. E. McKie and is the pGEM-3Zf(−) plasmid(Promega) into which has been cloned an HSV-1 fragment (123459-129403)consisting of the RL1 gene and its flanking sequences. The 477 bpPf7MI-BstEII fragment of the RL1 gene (125292-125769) has been removedand replaced with a multi-cloning site (MCS) to form RL1.del.

pNAT-IRES-GFP

** pNAT-IRES-GFP was supplied by Dr. Marie Boyd (CRUK BeatsonLaboratories) and is the pIRES2-EGFP plasmid (BD Biosciences Clontech)into which she has cloned the bovine noradrenaline transporter (NAT)gene (3.2 Kbp), at the NheI and XhoI sites.

***Transformation of Bacterial Cells

10 μl of a glycerol E. coli stock was added to 10 ml 2YT medium in a 20ml griener tube. This was placed in a 37° C. shaking incubator for 16-24hrs until a saturated culture was obtained. 1 ml of this culture wasthen added to 100 ml of 2YT in a 500 ml sterile glass bottle and placedin the 37° C. shaking incubator for 3 hrs. The bacterial cells werepelleted by centrifugation at 2,000 rpm for 10 minutes (Beckman). Thecells were then resuspended in 1/10th volume of transformation andstorage buffer (10 mM MgCl₂, 10 mM Mg(SO)₄, 10% (w/v) PEG 3,500, 5%(v/v) DMSO). The cells were placed on ice for between 10 minutes and 2hrs, after which time they were considered competent for transformation.

1-10 μl of DNA was mixed with 100 μl of competent bacteria in eppendorftubes, and the tubes placed on ice for 30 minutes. After this, thesamples were ‘heat shocked’ by incubating the tubes in a 42° C. waterbath for exactly 45 seconds before placing them on ice for a further 2minutes. 1 ml of L-Broth was added, the tube inverted 2-3 times, and thebacteria incubated for 1 hr at 37° C. 100 μl of the transformed bacteriawas plated out onto L-broth agar plates containing 100 μg/ml of theappropriate antibiotic (usually ampicillin or kanamycin). Plates wereallowed to dry at room temperature, before incubating in an invertedposition at 37° C. overnight.

Example 2 Generation of ICP34.5 null HSV-1 expressing a gene product ofinterest and GFP using plasmid RL1.dIRES-GFP.

General Approach

Generation of ICP34.5 null HSV-1 expressing a gene product of interestrequires insertion of nucleotide sequence encoding the gene product(polypeptide) of interest, and often a desired promoter, at the MCS ofRL1dIRES.GFP followed by co-transfection of BHK cells with thelinearised plasmid, containing the gene of interest, and HSV DNA.Following homologous recombination viral plaques expressing GFP areidentified. FIG. 7 illustrates the method steps involved.Referring to FIG. 7A plasmid DNA, containing the gene of interest andthe desired promoter (X), is digested with restriction endonucleases torelease the promoter/gene fragment.

The promoter/gene fragment is purified and cloned into the multi-cloningsite (MCS) of RL1.dIRES.GFP forming a shuttle vector suitable forgenerating oncolytic HSV-1 (FIG. 7B). This vector contains HSV-1sequences that flank the essential RL1 gene but does not contain the RL1gene. The plasmid also contains the gene for Green Fluorescent Protein(GFP) downstream of an internal ribosome entry site (IRES). The IRESpermits expression of both the gene of interest and the GFP gene fromthe same upstream promoter.

BHK cells are then co-transfected with linearised RL1.dIRES.GFP, nowcontaining the gene of interest, and HSV-1 DNA (FIG. 7C). Followinghomologous recombination, designer virus, expressing the gene ofinterest and GFP, is generated and can be distinguished from wild typevirus (also generated but not expressing GFP) under a fluorescencemicroscope.

Viral plaques, expressing GFP (and hence the gene of interest), arepicked under the fluorescence microscope and purified until allwild-type HSV-1 has been removed. The recombinant HSV-1 is considered100% pure when all the viral plaques are expressing GFP (FIG. 7D).

Once the recombinant virus is completely pure, an isolated plaque ispicked and a highly concentrated stock is grown and titrated (FIG. 7E).Oncolytic HSV-1, expressing a gene product of interest from a selectedpromoter, is then ready for characterisation and in vitro examination ofits tumour killing potential.

Materials and Methods

To generate recombinant ICP34.5 null HSV-1 expressing a gene of interestand GFP, requires the gene of interest, and often a suitable promoter,to be cloned into the MCS of RL1.dIRES-GFP in the forward orientationwith respect to the GFP gene in this plasmid. Once this has beenachieved the plasmid is linearised (i.e. digested with a restrictionenzyme that cuts only once, usually SspI or ScaI) in an irrelevantregion. 80% confluent BHK cells in 60 mm petri dishes are thenco-transfected with HSV-1 DNA and linearised plasmid DNA as describedbelow.

To generate replication restricted HSV-1, expressing the gene ofinterest and GFP, the gene of interest must be cloned into RL1.dIRES-GFPdownstream of a suitable promoter (e.g. CMV IE). The promoter isrequired upstream of the gene of interest for the production of abicistronic mRNA transcript. The IRES sequence between the two openreading frames in the transcript functions as a ribosome binding sitefor efficient cap-independent internal initiation of translation. Thedesign enables coupled transcription of both the gene of interest andGFP, followed by cap-dependent initiation of translation of the firstgene (gene of interest) and IRES-directed, cap-independent translationof GFP. Co-ordinate gene expression is thus ensured in thisconfiguration.

Co-Transfection of Virus and Plasmid DNA by CaPO₄ and DMSO Boost HSV-1(17⁺) DNA and 0.1-1 μg linearized SMART cassette containing the gene andpromoter of interest is pipetted into 1.5 ml eppendorf tubes containing1 μl of calf thymus DNA (10 μg/ml) and an appropriate volume ofdistilled water to give a final volume of 165 μl. The solutions are verygently mixed using a 200 μl pipette tip. 388 μl of HEBS, pH 7.5, (130 mMNaCl, 4.9 mM KCl, 1.6 mM Na₂HPO₄, 5.5 mM D-glucose, 21 mM HEPES) is thenadded, the solution mixed, before adding 26.5 μl of 2M CaCl₂ dropwiseand flicking the eppendorf tube two or three times. The samples are leftat room temperature for 10-15 minutes then added dropwise to 80%confluent BHK's in 60 mm petri dishes from which the medium has beenremoved. Following incubation at 37° C. for 45 minutes, the cells areoverlaid with 5 ml of ETC10 and incubated at 37° C. Three to four hourslater, the media is removed and the plates washed with ETC10. Forexactly 4 minutes, the cells are overlaid with 1 ml 25% (v/v) DMSO inHEBS at room temperature. After the 4 minutes, the cells are immediatelywashed three times with 5 ml ETC10 before overlaying with 5 ml of ETC10and returning to the incubator. The following day, fresh medium is addedto the cells. Two days later, when cpe is evident, cells are scrapedinto the medium, transferred to small bijoux and sonicated thoroughly.The sample is then stored at −70° C. until required (see section belowon plaque purification).

N.B. The volume of virus DNA to add is determined by undertaking theabove procedure without plasmid DNA, using a range of virus DNA volumesand choosing the volume that gives the greatest number of viral plaqueson the BHK monolayer after 2 or 3 days.

Plaque Purification

Sonicated samples from co-transfection plates are thawed and seriallydiluted 10 fold in ETC10. 100 μl from neat to the 105 dilution is platedout on confluent BHK's in 60 mm petri dishes from which the media hasbeen removed. After 45 minutes incubation at 37° C., the cells areoverlaid with 5 ml EMC10 and incubated at 37° C. for 48 hrs. The platesare then checked for the presence of viral plaques and those dishes withthe fewest, most separated plaques are placed under a fluorescentstereomicroscope. Recombinant virus, designed to express the greenfluorescent protein (GFP) in addition to the gene of interest, canclearly be distinguished from wild type virus using a GFP filter.Fluorescent plaques are picked using a 20 μl pipette and placed(including the tip) into an eppendorf tube containing 1 ml ETC10. Thesample is thoroughly sonicated before making serial 10 fold dilutions inETC10 and repeating the above purification procedure. The process isrepeated typically 3-4 times until every plaque on the BHK monolayer isfluorescent. Once this has been achieved, 50 μl of this sample is usedto infect BHK cells in roller bottles, in 50 ml ETC10, and a virus stockgrown.

Tissue Culture Media

BHK21/C13 cells are grown in Eagle's medium (Gibco) supplemented with10% newborn calf serum (Gibco) and 10% (v/v) tryptose phosphate broth.This is referred to as ETC10. For virus titrations and plaquepurification, EMC10 (Eagles medium containing 1.5% methylcellulose and10% newborn calf serum) is used to overlay the cells.

Example 3 Construction of HSV1716/CMV-NTR/GFP

General Approach

HSV1716/CMV-NTR/GFP was generated by cloning a 1.6 Kbp BamHI fragmentfrom pPS949¹⁰, consisting of the E. coli nitroreductase (NTR) genedownstream of the CMV IE promoter (pCMV), into the MCS of theRL1.dIRES-GFP smart cassette, in the forward orientation with respect tothe GFP gene in RL1.dIRES-GFP (FIG. 8). The resultant plasmid, namedRL1.dCMV-NTR-GFP, was then linearised and recombinant virus generatedand purified as described above. The plasmid pPS949 (referred to as‘pxLNC-ntr’ in Ref 10) containing the NTR gene downstream of the CMV IEpromoter (pCMV-NTR) in a pLNCX (Clontech) backbone, was a kind gift fromProfessor Lawrence Young, University of Birmingham, UK.

Materials and Methods

4×1 μg of pPS949 was digested with 10 units of BamHI (Promega), in asuitable volume of 10× buffer (Promega) and nuclease free water(Promega), at 37° C. for 16 hrs. The reaction mixture waselectrophoresed in a 1% agarose gel for 1 hr at 110 volts. The 1.6 KbpDNA fragment consisting of the CMV promoter upstream of the NTR gene(pCMV-NTR), was excised using a sterile scalpel and the DNA purifiedfrom the gel using a QIAquick Gel Extraction kit (Qiagen). 5 μl of thepurified DNA fragment was electrophoresed on a 1% agarose gel to checkits concentration (FIG. 9).

2 μg of the RL1.dIRES-GFP smart cassette was then digested with 15 unitsof BglII (Promega), in a suitable volume of 10× buffer (Promega) andnuclease free water (Promega), at 37° C. for 16 hrs. The digestedplasmid was then purified using the QIAquick PCR purification kit(Qiagen), treated with 10 units of Calf Intestinal Phosphatase(Promega), in a suitable volume of 10×CIP buffer and nuclease free waterfor 4 hrs at 37° C., before being purified again using the Qiaquick PCRpurification kit. 5 μl of the purified DNA was electrophoresed on a 1%agarose gel to check its concentration (FIG. 10).

Ligation reactions were carried out in small eppendorf tubes containing5 units T4 DNA Ligase (Promega), a suitable volume of 10×DNA LigaseBuffer (Promega), nuclease free water (Promega) and various volumes ofthe BglII digested/CIP treated RL1.dIRES-GFP smart cassette and pCMV-NTR(BamHI ends), at 16° C. overnight. Competent JM109 bacterial cells(Promega) were then transformed with various aliqouts of the ligationreactions. Colonies formed on the plates were picked, had their plasmidDNA extracted using a Qiagen Plasmid Mini kit and screened for insertsusing BglII/XhoI (Promega) restriction enzyme analysis. RL1.dIRES-GFPplasmid DNA containing the pCMV-NTR insert in the correct orientationwould produce two fragments of 11.5 Kbp and 300 bp following digestionwith BglII and XhoI. One clone (clone 4) was found to contain the insertin the correct orientation (FIG. 11). This plasmid was named‘RL1.dCMV-NTR-GFP’.

0.1-1 μg of RL1.dCMV-NTR-GFP was linearized by digesting with 10 unitsof ScaI (Promega), in a suitable volume of 10× buffer (Promega) andnuclease free water (Promega), at 37° C. for 16 hrs. A sample (5 μl) ofthe digested DNA was electrophoresed on a 1% agarose gel for 1 hr at 110volts to check that it had been linearized. 80% confluent BHK cells werethen co-transfected with a suitable volume of the remaining linearisedDNA and HSV-1 DNA. Recombinant HSV-1, expressing GFP (and hence NTR),was identified and purified using a fluorescent microscope and a virusstock, named HSV1716/CMV-NTR/GFP, was grown and titrated on BHK cells(FIG. 12).

HSV1716/CMV-NTR/GFP has been deposited in the name of CrusadeLaboratories Limited having an address at Department of NeurologySouthern General Hospital 1345 Govan Road Govan Glasgow G51 5TF Scotlandon 5 Nov. 2003 at the European Collection of Cell Cultures (ECACC),Health Protection Agency, Porton Down, Salisbury, Wiltshire, SP4 0JG,United Kingdom under accession number 03110501 in accordance with theprovisions of the Budapest Treaty.

HSV1716/CMV-NTR/GFP Cell Killing

HSV1716/CMV-NTR/GFP replicates with almost identical kinetics to HSV1716in BHK cells and 3T6 cells. BHK cells support the replication of ICP34.5null HSV while confluent 3T6 cells do not. FIG. 13 shows thatHSV1716/CMV-NTR/GFP will replicate as well as HSV1716 in permissive celllines and that the introduction of exogenous genes, e.g. NTR and GFP,has not reduced the oncolytic potential of the ICP34.5 null HSV. Thefact that HSV1716/CMV-NTR/GFP fails to replicate in 3T6 cells alsoindicates that this recombinant HSV is an ICP34.5 null mutant.

FIG. 14 is a Western blot demonstrating that no ICP34.5 polypeptide isexpressed from HSV1716/CMV-NTR/GFP, and that the virus is thus useful asa gene therapy vector.

FIG. 15 is another Western blot demonstrating expression of NTR in avariety of cell lines infected with HSV1716/CMV-NTR/GFP, including ahuman malignant melanoma cell line (C8161) and confluent 3T6 cells inwhich ICP34.5 null HSV does not replicate. Expression of NTR inconfluent 3T6 cells, following infection with HSV1716/CMV-NTR/GFP, isencouraging as it demonstrates that replication of this ICP34.5 nullmutant is not required for expression of the prodrug-activating gene(i.e. NTR). Some tumour cells in vivo will not support the replicationof ICP34.5 null HSV and as such, will not be killed with HSV1716.

FIG. 16 shows the results from a cytotoxicity assay performed inconfluent 3T6 cells. Infecting confluent 3T6 cells with an ICP34.5 nullmutant (HSV1716/CMV-NTR/GFP), at a multiplicity of infection (MOI) of 1plaque forming units (pfu)/cell, does not result in any significant celldeath, neither does separate incubation of the cells with 50 μM CB1954.However, significant cell death is evident 72 hrs post infection with 1pfu/cell HSV1716/CMV-NTR/GFP when 50 μM CB1954 is included in the growthmedium. This clearly demonstrates that when there is no replication ofthe virus, substantial cell death is still possible from virus directedenzyme prodrug therapy (VDEPT).

Infecting confluent 3T6 cells with an ICP34.5 null mutant at a MOI of 10pfu/cell will result in cell death, by a mechanism known as ‘viralantigen overload’. However, the level of cell killing is even morepronounced (approximately 20% more), when 50 μM CB1954 is included inthe growth medium.

A similar cytotoxicity assay was performed in human C8161 melanomacells, the results are set out in FIG. 17. Unlike confluent 3T6 cells,C8161 cells do support the replication of ICP34.5 null HSV. Therefore,cell death will occur following infection of the cells with ICP34.5 nullHSV, at 1 pfu/cell. However, when CB1954 is included in the overlay ofHSV1716/CMV-NTR/GFP infected cells, the cells are killed moreefficiently and more quickly. No enhanced cell killing is evident whenCB1954 is included in the overlay of cells infected with HSV1716-GFP.These results demonstrate that enhanced cell killing is possible inhuman tumour cells.

Cell culture images for the cytotoxicity assays performed in confluent3T6 and human C8161 melanoma cells are shown in FIGS. 18 and 19.

Example 4

In vivo evaluation of the anti-tumour activity of a selectivelyreplication competent herpes simplex virus in combination with enzymepro-drug therapy. The anti-tumour activity of a selectively replicationcompetent herpes simplex virus in combination with an enzyme prodrugtherapy approach in appropriate animal models in vivo was investigated.

The parental virus, HSV1716 is a selectively replication competentmutant of the herpes simplex virus 1 (HSV1) which lacks both copies ofthe RL1 gene that encodes the protein ICP 34.5. This protein is aspecific determinant of virulence. The function of this protein has beendescribed at length elsewhere¹². The virus can grow only in cells thathave a high level of functional PCNA. High levels of PCNA are found onlyin cells that are dividing such as tumour cells and not normaldifferentiated cells.

It has already been shown that HSV1716 can achieve selective tumour cellkilling with minimal toxicity and improved survival times in a number ofanimal models¹³. Initial phase 1 clinical trials using HSV1716 virus inpatients has also meet with some success^(14,15).

Although HSV1716 selectivity replicates in tumour reducing the tumourbulk by cell lysis the inventors did not anticipate HSV1716 to lyticallyreplicate in all cells in the tumour due to the heterogeneity of thecell type and growth state.

In order to enhance the efficacy of the tumour cell killing—hence killthe entire tumour—the inventors have constructed a derivative of HSV1716designated HSV1716/CMV-NTR/GFP that expresses the E. Coli nitroreductasegene (ntr) under the control of a CMV early promoter (see example 3above). In this example and the figures referred to HSV1716/CMV-NTR/GFPis called HSV1790.

The enzyme ntr converts the inactive prodrug CB1954 to a functionalcytotoxic alkylating agent that kills both dividing and non-dividingcells by apoptosis. This active drug is diffusible and membranepermeable resulting in an efficient bystander effect, i.e. wherein theactivated drug may have an effect on surrounding cells.

As the prodrug will only be converted to its active form in the tumourwhich has been infected with ntr expressing virus, toxicity to normalcells is avoided hence improving the therapeutic index followingsystemic delivery of this compound.

Initial in vitro experiments using this combination have already shownenhanced cell kill using this virus in combination with CB1954 in anumber of cell lines.

This example further evaluates this combination approach in vivo inappropriate animal models.

Results

Months 1-3

Months 1-3 were taken up mainly by in vitro work. During this timeperiod high titre, sterile virus stock was generated for use in thexenograft models.

Xenograft models were also generated in athymic nude mice using the cellline A2780, a human ovarian epithelial carcinoma line initially derivedfrom a tumour sample from an untreated patient (European Collection ofCell Cultures (ECACC) CAMR, Porton Down, Salisbury, Wiltshire, SP4 0JG,United Kingdom, accession number 93112520).

Generation of a gliomal xenograft model was attempted using 2 gliomallines that were available in house, LN-18 and U373MG. There are reportsin the literature of both being successfully grown as xenografts inathymic mice. However, as shown in the table below the inventors failedto see any xenograft growth up to 28 days after injection with 5 millioncells subcutaneously.

TABLE 1 Presence of No of cells injected xenograft 28 days Cell Line permice Number of mice after cell injection LN 18 5 million 5 0/5 U373 MG 5million 5 0/5

A2780 Tumour Take

As reported previously A2780 have a Take Rate of approximately 50%—thatis 50% of mice that are injected with 5 million cells per flanksubcutaneously will develop xenografts. When the number of cellsinjected was increased to 10 million or more an increase in take rate ofapproximately 15-25% was seen, giving an overall take rate of 65-75%.

Thus increasing the number of gliomal cells injected may increase thetake rate of these cell lines

Dose Response to the HSV1790 Virus

Before the mice can be treated with a combination of virus and prodrug,first one must carry out experiments to make informed decisions abouthow much of the virus, and the prodrug to give.

A dose response experiment will allow one to find both the mostappropriate does of the virus to use in the experiments and the maximumtolerated dose (MTD) of the virus, that is, the largest amount of thevirus that can be given to a single mouse without adverse side effects.Small groups of tumour bearing mice are given a small dose of virus.Assuming they do not have any adverse effects another group is given alarger dose of virus. This continues until either the mice start tosuffer ill effects or we reach a maximum dose.

The maximum amount of virus that can be intratumourally injected is 100μl, hence the maximum dose from our current stock is 10⁹ PFU perinjection.

FIG. 20 shows the weight change in the mice after injection with avariety of doses of virus. Weight is a good indicator of the animalsoverall health. Any loss of weight signifies that the treatment is notbeing well tolerated. Where an animal loses more than 20% of its initialbody weight it was sacrificed immediately.

A dose of 10⁹ PFU of the HSV1790 virus is not tolerated by these mice,they rapidly lost body weight and were sacrificed at Day 3 postinjection. Doses of 10⁸ PFU or less were better tolerated, the miceinitially lost weight in the days following injection but quicklyrecovered to approximately their initial body weights.

It should be pointed out that as the experiment progresses the animalsappear to be increasing in weight. This is almost certainly due to thefact that it is total body weight that is measured, which includes theweight of any tumour that is forming.

Response of the Tumour to HSV1790 Treatment

Tumour volume was measured daily after intratumoural injection of theHSV1790 virus to look for any growth delay or regression of the tumours.

FIG. 21 shows the change in tumour volume as measured over a period of100 days. If the tumour was injected with PBS only as a control thetumour increased in size rapidly and by approximately Day 13 postinjection the tumours had become too large and the animals had to besacrificed.

Treatment with all doses of virus appeared to delay the growth of thetumour to some degree. Doses of 10⁵ PFU increased the longevity of themice by approximately 12 days while mice injected with 10⁶ PFU virustumours survived for an extra 23 days compared to the control groupbefore the tumours became prohibitively large. Perhaps surprisingly thegroup of mice injected with 10⁸ PFU of virus survived only slightlylonger than the control group. It is possible there were a large numberof non infectious particles or that sheer number of particles caused thecells which the virus would have grown in to be killed.

The group of mice treated with 10⁷ PFU of virus survived the longest andindeed two out of three of the mice did not have any visible signs oftumour when sacrificed at day 100.

Naked DNA Experiments

In order to check the alterations in tumour growth are due to the virusitself and not a result of the CMV-ntr plasmid DNA that had beenintroduced to the HSV1716 virus, an experiment was set up looking at theeffect of the CMV-ntr plasmid DNA alone and in combination with theprodrug CB1954.

Mice were randomised into treatment groups of 6 animals each when tumourdiameters are approximately 5 mm (this is Day 0). FIG. 22 shows thestarting tumour diameters for the mice used in this experiment. Twogroups of mice were administered CMV-ntr plasmid by direct intratumouralinjection at a dose of 0.2 mg DNA per injection. One of these groups wasthen administered with a single dose of 80 mg/kg of CB1954 on Day 2 byintra-peritoneal injection. The third group of mice had a singleadministration of CB1954 (80 mg/kg) by intra-peritoneal injection on Day2 following intratumoural injection of saline control at Day 0. Animalswere weighed daily (FIG. 23) and daily caliper measurements performeduntil the tumour sizes were in the region of 20 mm by 20 mm. Tumourvolumes were estimated from these measurements (volume=d3×6) (FIG. 24).In addition any toxicity from these administered agents was determined.On the basis of these experiments the inventors determined that neitherthe CMV-ntr alone, CB1954 alone or the combination of both CMV-ntr andCB1954, has any anti-tumour activity as determined by tumour regressionin this model system (FIG. 24).

Scheduling Experiment

Previous dose response experiments have shown that doses of less than10⁸ PFU virus per mouse do not appear to have any adverse effect of theanimals health.

A dose of 10⁷ PFU virus per mouse resulted in a great reduction intumour growth, indeed after 100 days two out of three of the mice in thegroup had no visible tumour. This is very encouraging—the virus only athigh doses may be enough to delay growth or cause tumour regression.

However to look at the effect of a combination of the virus and theprodrug CB 1954 a lower dose of the virus was studied—if the treatmentof the virus alone results in growth delay for such a long period onewould be unable to ascertain the addition or synergistic effects of theprodrug.

The inventors proceeded to investigate two doses of the virus incombination with CB1954. The doses selected were 10⁵ PFU and 10⁶ PFU.Both these doses caused some tumour growth delay in earlier experiments.

The prodrug is given as an 80 mg/kg intra-peritoneal injection, afterdissolving the powdered form in 10% acetone and then making up thevolume with peanut oil.

Another factor that determines how well the drug will work—hence howmuch tumour growth delay or regression is seen—is when the drug isactually given. As the prodrug will only be converted to an activesubstrate in the presence of NTR it was considered that the viruscontaining the NTR will have to been given first. It was also consideredthat if the virus is given time to replicate and produce more NTR thenthe prodrug may have a more pronounced effect.

To discover the optimal doses of both the virus and the drug and theoptimal times of these treatments a scheduling experiment was set up.

Mice were randomized into treatment groups (treatment regimes shown inTable 2) of 3 animals when tumour diameters were approximately 5 mm(tumour volume 0.5-1.5 mm³). FIG. 25 shows the starting tumour volumesof each of the groups.

TABLE 2 Treatment groups 1 10⁵ HSV 1790 + drug (Day 2) + Drug (Day 10) 210⁵ HSV 1790 + drug (Day 2) + vehicle (Day 10) 3 10⁵ HSV 1790 + vehicle(Day 2) + drug (Day 10) 4 10⁵ HSV 1790 + vehicle (Day 2) + vehicle (Day10) 5 No virus + drug (Day 2) + drug (Day 10) 6 No virus + drug (Day2) + vehicle (Day 10) 7 No virus + vehicle (Day 2) + drug (Day 10) 8 Novirus + vehicle (Day 2) + vehicle (Day 10) 9 10⁶ HSV 1790 + vehicle (Day2) + vehicle (Day 10) 10 10⁶ HSV 1790 + drug (Day 2) + vehicle (Day 10)11 10⁶ HSV 1790 + drug (Day 2) + drug (Day 10) 12 10⁶ HSV 1790 + drug(Day 2) + drug (Day 10) + drug (Day 15) 13 No virus + drug (Day 2) +drug (Day 10) 14 No virus + vehicle (Day 2) + vehicle (Day 10) 15 10⁵HSV 1716 + drug (Day 2) + drug (Day 10) 16 10⁵ HSV 1716 + vehicle (Day2) + vehicle (Day 10) 17 10⁵ HSV 1716 + drug (Day 2) + vehicle (Day 10)18 10⁵ HSV 1716 + vehicle (Day 2) + drug (Day 10)

The treatment groups were administered with a single directintratumoural injection of the virus and dose determined for that group.The virus was diluted PBS+10% serum. ‘No virus’ control groups receivedan intratumoural injection of 100 μl of PBS+10% serum. This day wasdesignated as Experimental Day 0.

The intratumoural injections did not appear to have any adverse effectson the mice. Some tumours bleed slightly after injection but not to agreat degree. The animals did not lose body weight (FIG. 26) and theirbehaviour did not appear to alter. In all the tumours that bleedslightly, the following day the healing process had begun and within 3-5days there was little evidence of any puncture wound on any tumour.

Injections of CB1954 were given to the appropriate groups at days 2, 10and 15. A dose of 80 mg/kg—the equivalent of approx. 2 mg per mouse—wasgiven. The powdered form of the CB1954 drug (from Sigma) was dissolvedin acetone to 10% of the final volume (10 μl per 2 mg). The volume wasthen made up to 2 mg CB1954 in 100 μl using peanut oil. A syringe wasused to mix the drug as peanut oil is thick and viscous. The drug wasmade up fresh every time.

The appropriate groups were then injected intra-peritoneal with thissolution. Control groups which were not receiving drug were injectedintra-peritoneal with a 100 μl solution of 10% acetone in peanut oil.

No swelling or irritation at the site of injection was noted on any ofthe mice either at time of injection or at any later time point. Themice appeared slightly lethargic for a short period after the injectionbut did not lose any body weight (FIG. 26) or show signs of lethargy thefollowing day.

No virus+CB1954 prodrug

Groups 5, 6, 7, 8, 13 & 14 looked at the effect of prodrug alone ontumour growth. FIG. 27 shows that there is little effect on tumourgrowth when CB1954 is given alone.

10⁵ PFU Virus+/−CB1954 Prodrug

10⁵ PFU virus was given at Day 0 followed by either prodrug or vehicleat Days 2 and 10.As can be seen from the graph in FIG. 28 tumours treated with eithervirus only or virus and prodrug did not grow as large as the untreatedtumour. The tumour treated with the virus grew only to approximatelyhalf the size of the untreated control.

Treatment with virus and prodrug resulted in tumours which grew to onlyapprox 2-3 mm in volume. This is significantly less than the untreatedtumours which grew in size to approx 20 mm³

10⁶ PFU Virus+/−CB1954 Prodrug

FIG. 29 shows the changes in tumour volume over time after treatmentwith a higher dose of virus, 10⁶ PFU per injection, in combination withthe prodrug, given as described in Table 2. As with the lower virusdose, treatment with either virus only, or in combination with CB1954,results in significantly smaller tumours compared to the untreatedcontrols.

HSV1716 Virus in Combination with CB1954 Prodrug

The parental strain of the virus, which has not been engineered tocontain the CMV-ntr DNA was examined for its effects on tumour growthdelay. This virus does have an oncolytic effect, however it doesn'tcontain the NTR gene needed to convert the inactive prodrug into itsactive metabolite. Therefore one would not expect any additional orsynergistic effects when the prodrug is added in combination with thevirus. FIG. 30 shows the results of this experiment.

The combination of the virus and the prodrug appeared to produce somereduction in tumour growth compared to the untreated control tumours.

The groups used in these results contained only 2 or 3 animals. Theanimals used were also older and their tumours had taken longer to growthan those used in previous experiments. Hence it is possible thatrepeating the experiment with a larger number, with younger mice orquicker forming tumours may result in a more marked growth delay aftertreatment with the HSV1716 virus.

Comparison of HSV1790 (at 10⁵ and 10⁶) and HSV1716 in Combination withCB1954 Prodrug

FIG. 31 shows a comparison between the two doses of the HSV1790 virus incombination with the prodrug and the HSV1716 prodrug combination. Theparental virus HSV1716 shows some growth delay in comparison with theuntreated control. We would assume that this growth delay is due to theoncolytic effect of the virus as the NTR gene is not present to alterthe inactive prodrug into its active form.

Tumour growth is reduced further when the tumour is treated with the HSV1790 virus containing the NTR gene. This appears to be dosedependent—the higher dose of the virus results in a greater growth delaythan the lower dose.

In conclusion it would appear from these results that indeed the HSV1790virus used in combination with the prodrug CB1954 results in growthdelay in the model system examined. Giving both virus and drug incombination has a greater effect than given either alone.

It appears that the timing at which the prodrug is given after virustreatment is important. When CB1954 was given soon after viral injection(Day 2 post viral injection) tumour growth was not delayed as much as ifthe drug was given at a later date (Day 10). It may be that given at Day2 the drug killed the cells that were supporting viral growth andreplication and actually reduced the oncolytic effect of the virus.

By day 10 the virus may have replicated and killed as many cells byoncolysis as possible. It is anticipated that due to heterogeneity ofthe cell type and growth state that all the cells within a tumour wouldnot be susceptible to lysis by the virus. The drug then comes in and‘mops up’ by killing any cells that are supporting viral growth (hencecontaining the NTR gene) but were not susceptible to oncolysis. As theactive drug is diffusible and membrane permeable it may have a bystandereffect—killing not only the cells infected with the virus but also itsnear neighbours.

REFERENCES

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1. An oncolytic herpes simplex virus wherein the herpes simplex virusgenome comprises nucleic acid encoding an heterologous nitroreductase(NTR), wherein the genome has an inactivating mutation in the RL1 locussuch that the herpes simplex virus does not produce a functional ICP34.5gene product, and wherein the herpes simplex virus is a variant of oneof HSV-1 strains 17 or F or HSV-2 strain HG52.
 2. The herpes simplexvirus of claim 1, wherein the herpes simplex virus genome comprisesnucleic acid encoding an heterologous NTR, wherein the genome has aninactivating mutation in the RL1 locus such that the herpes simplexvirus does not produce a functional ICP34.5 gene product, and whereinthe genome of the herpes simplex virus otherwise is the genome of HSV-1strain 17 or F or HSV-2 strain HG52.
 3. The herpes simplex virus ofclaim 1, wherein the herpes simplex virus genome comprises a nucleicacid encoding an heterologous NTR, wherein the herpes simplex virusgenome has an inactivating mutation in the RL1 locus such that theherpes simplex virus does not produce a functional ICP34.5 gene product,wherein the herpes simplex virus genome has a mutation in theribonucleotide reductase gene, and wherein the herpes simplex genomeotherwise is the genome of HSV-1 strain 17 or F or HSV-2 strain HG52. 4.The herpes simplex virus of claim 1, wherein the herpes simplex virus isa variant of one of HSV-1 strains 17 or F.
 5. The herpes simplex virusof claim 1 wherein the nitroreductase encoding nucleic acid comprisesSEQ ID NO: 2, or a nucleic acid encoding the polypeptide of SEQ ID NO:1, or a nucleic acid having at least 90% sequence identity to SEQ ID NO:2 or to a nucleic acid encoding the polypeptide of SEQ ID NO:
 1. 6. Amethod for the treatment of a tumour involving lysing or killing tumourcells in a patient in need of treatment, the method comprising the stepsof (i) administering to the patient the herpes simplex virus of claim 1,and (ii) administering to said patient a therapeutically effectiveamount of an NTR prodrug.
 7. The herpes simplex virus of claim 1 whereinsaid herpes simplex virus genome further comprises a regulatorynucleotide sequence operably linked to said nucleic acid encoding NTR,wherein said regulatory nucleotide sequence has a role in controllingtranscription of said NTR.
 8. The herpes simplex virus of claim 1wherein said nucleic acid is located in at least one RL1 locus of theherpes simplex virus genome.
 9. The herpes simplex virus of claim 1wherein said nucleic acid is located in, or overlaps, at least one ofthe ICP34.5 protein coding sequences of the herpes simplex virus genome.10. The herpes simplex virus of claim 1 wherein the herpes simplex virusis a variant of HSV-1 strain 17 mutant
 1716. 11. The herpes simplexvirus of claim 1 in which all copies of the ICP34.5 gene present in theherpes simplex virus genome are disrupted such that the herpes simplexvirus is incapable of producing a functional ICP34.5 gene product. 12.The herpes simplex virus of claim 1 which lacks only one expressibleICP34.5 gene.
 13. The herpes simplex virus of claim 1 wherein saidnucleic acid encoding the heterologous nitroreductase (NTR) forms partof a nucleic acid cassette integrated in the genome of said herpessimplex virus, said cassette encoding: (a) said nucleic acid encodingNTR; and nucleic acid encoding (b) a ribosome binding site; and (c) amarker, wherein the nucleic acid encoding NTR is arranged upstream (5′)of the ribosome binding site and the ribosome binding site is arrangedupstream (5′) of the marker.
 14. A medicament, pharmaceuticalcomposition or vaccine comprising the herpes simplex virus of claim 1.15. A composition comprising the herpes simplex virus of claim 1 and anNTR prodrug.
 16. A composition comprising the herpes simplex virus ofclaim 1 and CB1954.
 17. A kit of parts comprising a first containerhaving a quantity of the herpes simplex virus of claim 1 and a secondcontainer having a quantity of an NTR prodrug.
 18. A method ofexpressing in vitro or in vivo a nitroreductase, said method comprisingthe step of infecting at least one cell or tissue of interest with theherpes simplex virus of claim 1, wherein the genome of said viruscomprises a nucleic acid sequence encoding an heterologousnitroreductase in at least one of the long repeat regions (R_(L)), saidnitroreductase operably linked to a transcription regulatory sequence.19. A method for the treatment of a tumour comprising the steps of: (i)administering to a patient in need of treatment a therapeuticallyeffective amount of an oncolytic herpes simplex virus, wherein thegenome of said virus comprises a nucleic acid sequence encoding anheterologous nitroreductase (NTR) in at least one of the long repeatregions (R_(L)), and wherein the genome has an inactivating mutation inthe RL1 locus such that the herpes simplex virus does not produce afunctional ICP34.5 gene product, and wherein the herpes simplex virus isa variant of one of HSV-1 strains 17 or F or HSV-2 strain HG52; and (ii)administering to said patient a therapeutically effective amount of anNTR prodrug.
 20. An oncolytic herpes simplex virus, wherein the genomeof said virus comprises a nucleic acid sequence encoding an heterologousnitroreductase (NTR) in at least one of the long repeat regions (R_(L)),wherein the genome has an inactivating mutation in the RL1 locus suchthat the herpes simplex virus does not produce a functional ICP34.5 geneproduct, and wherein the herpes simplex virus is a variant of one ofHSV-1 strains 17.